Mehmet R. Dokmeci Harvard Medical School
Brigham and Women's Hospital
Junji Fukuda University of Tsukuba
Ali Khademhosseini Harvard-MIT Division of Health Sciences and Technology
Hirokazu Kaji Tohoku University
II3: Poster Session
Monday PM, November 28, 2011
Exhibition Hall C (Hynes)
1:00 AM -
II3.2 TRANSFERRED TO II8.10
1:00 AM -
II3.3 TRANSFERRED TO II1.8
1:00 AM -
II3.5 Transferred to II2.2
II1: Microfluidics for Cellular Microenvironments I
Monday AM, November 28, 2011
Room 206 (Hynes)
9:30 AM - **II1.1
Microfluidic Technology for Building and Handling 3D Tissue Structures.
Shoji Takeuchi 1 2 Show Abstract
1 , Univ. of Tokyo, Tokyo Japan, 2 , JST ERATO, Tokyo Japan
In this presentation, I will talk about several approaches for 3D tissue construction based-on MEMS/Microfluidic technology. We demonstrated a 3D tissue structure by stacking the "cellular" beads into a 3D mold. Since this method allows us to fabricate 3D in vivo like tissue structures, it can be applicable in the fields of regenerative medicine and drug development. To prepare the cellular beads, we used an axisymmetric flow focusing device (AFFD) that allows us to encapsulate HepG2 cells within monodisperse collagen beads. We then seeded 3T3 cells on the surface of the collagen beads. Finally HepG2 and 3T3 cells were successfully made contact with each other. Moreover, by putting these capsules in a 3D chamber and incubating them, we successfully established complicated and milli sized 3D structures. We believe that altering the shape can be possible as simple as changing the mold, and will try to combine multiple types of cells to create more complex system that functions as a living organism.
10:00 AM - II1.2
A Polymeric 3D Artificial Compound Eye for Wide-Angle Imaging.
Hansong Zeng 1 , Yi Zhao 1 Show Abstract
1 Biomedical Engineering Department, Ohio State University, Columbus, Ohio, United States
This abstract reports a bio-inspired artificial “compound eye”, which can view optical images with wide field-of-view. The technology is achieved by a smart microfluidic configuration, which uses cost-effective materials and simple fabrication. Because of the unique features, this device can potentially be applied in areas including endoscopic visualization, military surveillance, environment monitoring, consumer electronics and so forth .Biological research reveals that mammalian animals and insects have distinct vision mechanisms . The former usually have a pair of camera eyes whose focal length can be adjusted to obtain a high definition image; while the latter usually have compound eyes that have a wide field-of-view but poor resolution. In this work, an engineering solution that integrates features of both mammalian animals’ and insects’ eyes is realized using an opto-microfluidic system.The structure is realized by two layers of microfluidic channel network. In the first layer, an array of circular membranes is deployed at the close end of the channels. The refractive liquid medium is supplied to actuate the membranes and form the lenses. The curvature and hereby the focal length of each lens is adjusted by the pumping pressure. In the second layer, another medium with different optical refractive property is applied to create the hemispherical dome. It also steer the lenses to orient in different directions. The device is fabricated using standard soft lithography process. The pressure is controlled by external pressure sources in this work. The focal length of an individual lens can be tuned from the infinity to less than 1mm with a small pressure change, suggesting that an integration of micro-pumps into the system is possible in our future work. The prototype of a 3D artificial compound eye is fabricated. The images acquired clearly show that each lens behaves as an individual camera eye and the multiple images form a comprehensive view around the optical device.In conclusion, an advanced 3D artificial compound eye with focus-tunable lenses is successfully developed using opto-microfluidic technology. The optical device is able to present images with wide field-of-view and high definition. It mimics the structures of both insects’ compound eyes and mammalian animals’ camera eyes and enjoys the advantages of both.ReferenceW. Sturzl, et al, "Mimicking honeybee eyes with a 280 degree field of view catadioptric imaging system," Bioinspiration & Biomimetics, vol. 5, 2010.J. W. Duparre et al., "Micro-optical artificial compound eyes," Bioinspiration & Biomimetics, vol. 1, 2006.
10:15 AM - II1.3
Hybrid Silicon MEMS/Biogenic Silica Microfluidics Platform for Separating and Detecting Transport of Ions and Molecules.
Kai-Chun Lin 1 , B. Ramakrishna 1 , Xiaofeng Wang 2 , Michael Goryll 2 Show Abstract
1 School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, Arizona, United States, 2 School of Electrical, Computer and Energy Engineering, Arizona State University, Tempe, Arizona, United States
Micro- and nanofluidic technology offers not only the potential to dramatically reduce sample volume and enhance the speed of fluidic separation and (bio-) chemical detection, but also to selectively perturb locations on a micro/nanostructure. Top-down design of nanofluidic devices, however, is still a challenging endeavor. Biogenic silica nanostructures, derived from diatoms, possess highly ordered porous hierarchical nanostructures. The complex 3D arrays of silica pores have very unique properties, making it the ultimate nanofluidic device to select and capture molecules and colloids. In this research, we exploit the unique properties of Coscinodiscus wailesii - a species with a fairly large structure (~200 microns in diameter), that exhibits a narrow size distribution of nanopores on the order of 40 nm in diameter. We will present how a top-down/bottom-up approach can be used to integrate biogenic silica nanostructures with micromachined silicon substrates to create micro/nano hybrid systems and devices. Results from the study of transport phenomena will be presented for ions, such as hydronium, potassium and sodium, as well as polystyrene nanobeads gold nanoparticles and drug molecules of interest. We expect to leverage the understanding of the fluidic transport phenomena through well-characterized hierarchical pore structures towards designing hybrid devices for applications in separation and sensor technologies. Initial results will be presented on addressing single cells that are cultured on the surface of these hybrid structures.
10:30 AM - **II1.4
Neuroscience on a Chip.
Albert Folch 1 Show Abstract
1 Bioengineering, University of Washington, Seattle, Washington, United States
Cell culture technology is falling behind in the pace of progress. As animal and bacterial genomes and proteomes are being fully probed with DNA chips and a wide array of analytical techniques, a picture of cells with dauntingly complex inner workings is emerging. Yet cell culture methodology has remained basically unchanged for almost a century: it consists essentially of the immersion of a large population of cells in a homogeneous fluid medium. This approach is becoming increasingly expensive to scale up and cannot mimic the rich biochemical and biophysical complexity of the cellular microenvironment.Microtechnology offers the attractive possibility of modulating the microenvironment of single cells and, for the same price, obtain data at high throughput for a small cost. Microfluidic or “Lab on a Chip” devices, in particular, promise to play a key role for several reasons: 1) the dimensions of microchannels can be comparable to or smaller than a single cell; 2) the unique physicochemical behavior of liquids confined to microenvironments enables new strategies for delivering compounds to cells on a subcellular level; 3) the devices consume small quantities of precious/hazardous reagents (thus reducing cost of operation/disposal); and 4) they can be mass-produced in low-cost, portable units. Not surprisingly, in recent years there has been an eruption of microfluidic implementations of a variety of traditional bioanalysis techniques. I will review the latest efforts of our laboratory in the development of cell-based microdevices for neurobiology studies, such as neuromuscular synaptogenesis, axon guidance, and olfaction.
11:30 AM - **II1.5
Multipurpose Microfluidic Probes: Dipoles, Quadrupoles and Electrochemical Sensors for Studies with Cells and Tissue.
David Juncker 1 Show Abstract
1 Biomedical Engineering Department, McGill University, Montreal, Quebec, Canada
Microfluidic probes have been proposed for local perfusion and processing of surfaces, cells and tissues. Here, we will present our recent work in this area and present data on perfusion of organotypic slices using dipolar probes with one injection and one aspiration aperture. Next, we will present theoretical analysis of dipolar and quadrupolar microfluidic probes along with experimental validation. We introduce the concept of floating gradient that is formed at the stagnation point of a microfluidic probe and show that it can be rapidly tuned in space and time yet with minimal shear stress. We will discuss the integration of electrodes into the probes for electrochemical sensing of proteins by the probe. Challenges and future work will conclude this presentation.
12:00 PM - **II1.6
Microfluidic Platforms for Study of 3D Cell Chemotaxis within Biomaterials.
Amir Shamloo 2 , Sarah Heilshorn 1 Show Abstract
2 Mechanical Engineering, Stanford University, Stanford, California, United States, 1 Materials Science & Engineering, Stanford University, Stanford, California, United States
Chemotaxis, the directed migration of cells in response to a soluble biochemical gradient, is a critical process that directs cell movement during embryonic development, adult tissue remodeling, and cancer progression. Traditional in vitro chemotaxis assays (e.g., Boyden chambers and transwells) have a number of severe limitations, despite being widely used within the cell biology community. These traditional assays are not conducive to direct cell imaging, are generally difficult to quantify theoretically, do not produce stable concentration gradients, and cannot be adapted to test chemotaxis within 3D biomaterials. In response to these limitations, several groups have developed the use of microfluidic gradient generators to study cell chemotaxis. Microfluidics have several advantages for chemotactic studies, including ease in fabrication, low consumption of costly reagents, the capability to perform parallel experiments on a single chip, and the ability to predict and fabricate equilibrium concentration profiles of soluble cues. Building on this work, we have designed a microfluidic chemotactic generator that enables real-time visualization of chemotaxis and collective cell migration within 3D biomaterials. These microfluidic platforms are being used to study the biomechanical and biochemical factors that regulate endothelial cell movement during sprouting morphogenesis. Endothelial cell sprouting is a critical early step in angiogenesis, the formation of new blood vessels from existing conduits. Using this platform, we have identified that the G-protein coupled receptor 124 (GPR124) is a previously unknown regulator of blood vessel development in the brain. Furthermore, we have used these devices to screen various biomaterial formulations for their ability to induce stable endothelial sprouting upon exposure to vascular endothelial growth factor (VEGF) gradients. Intriguingly, our experiments find that endothelial sprouts alter their sensitivity to VEGF depending on the matrix density, suggesting a complex interplay between biochemical and biomechanical factors. As matrix density increases, steeper VEGF gradients and higher VEGF absolute concentrations are required to induce directional sprouting. In lower density matrices, endothelial sprouts were frequently observed to change their direction of growth by turning to reorient parallel to the VEGF gradient, a behavior reminiscent of the path-finding behavior of neuronal axons. In contrast, in higher density matrices this turning phenomenon was only rarely observed. Together, these results suggest new anti-angiogenic strategies for potential cancer treatment as well as pro-angiogenic strategies for regenerative medicine scaffolds.
12:30 PM - II1.7
A Microfluidic Assay for Measuring Electrical Conductivity of Gap Junction Channels.
Cedric Bathany 1 , Derek Beahm 2 , Steve Besch 2 , Frederick Sachs 2 , Susan Hua 1 2 Show Abstract
1 Dept. of Mechanical & Aerospace Eng. , SUNY Buffalo, Buffalo, New York, United States, 2 Department of Physiology and Biophysics, SUNY-Buffalo, Buffalo, New York, United States
Gap Junction Channels are the transmembrane protein structures that connect neighboring cells and responsible for the intercellular exchange of ions and metabolites. Most cells are known to express multiple connexins that form different types of junctions, and unfortunately, there are no known blocking reagents for a specific type of junction channel. We have previously developed a microfluidic based assay capable of measuring gap-junction mediated dye diffusion in cultured cells. In this paper, we present a microfluidic chip that measures the conductance across gap junction channels in real time. The chip contains tri-stream laminar flow across a 2D cell array. Two platinum electrodes are located under each side-stream forming a four-point resistance measurement. The middle stream contains sucrose solution that creates a non-conducting fluid barrier, known classically as the sucrose gap. Thus, when current is passed from one side of the gap to the other, it can only flow through the cells. The microfluidic channel was constructed using SU-8 on Pyrex glass substrate and Platinum electrodes deposited by sputtering deposition. Numerical simulation and empty channel calibration experiments were conducted to characterize the performance of the chip in terms of multiple flow control, interface stability, and fluid exchange time. Using this sensor device we tested the effect of the gap junction blocker, 2-APB, on Cx43 gap junctions in Normal Rat Kidney (NRK) cells. The results show that 2-APB reversibly inhibits Cx43 gap junction channels, and the blockage is dose dependent. The conductance was reduced by 13 kΩ and 63 kΩ in the presence of 100 and 200 µM of 2-APB, respectively, compared with control experiment. The time course of impedance change was fit using Boltzmann equation and it was found that the half saturation of the inhibition and recovery of 2-APB was approximate 51±10 sec. This chip allows us to measure conductance and molecular diffusion across gap junction channels simultaneously. Using the same chip, we have simultaneously measured the diffusion of 5-(and-6)-Carboxyfluorescein Diacetate, AM (5(6)-CFDA) and the conductivity through Cx43 in the presence of 2-APB. The results show 2-APB inhibits both electrical and diffusion coupling of gap junction channels in NRK cells with similar kinetics. The presented work demonstrated that our microfluidic sensor provides a generic platform for screening pharmacological agents, and the kinetics of the inhibitors on electrical conductance and molecular diffusion can be compared.
12:45 PM - II1.8
Method for Efficient Droplet Extraction from Covered Droplet-in-Oil Electrowetting-on-Dielectric Devices.
Haig Norian 1 , Ioannis Kymissis 1 , Kenneth Shepard 1 Show Abstract
1 , Columbia University, New York, New York, United States
Electrowetting is the phenomenon in which a polarizable liquid droplet undergoes a reduction in contact angle in the presence of an applied electric field. By selectively assigning voltages to an array of metal electrodes coated with a hydrophobic dielectric, we can facilitate droplet transport down to the picoliter scale, enabling a true lab-on-chip without the use of bulky mechanical pumps common in channel-based microfluidic devices. Due to the rapid evaporation of such small volumes of liquid, the typical electrowetting lab-on-chip configuration consists of the analyte/reagent droplets immersed in an oil, often dodecane, and sealed with a coverslip. Extraction of the droplet from the covered oil region into oil-free uncovered configuration has been analyzed in our laboratory setup. We propose the use copper perfluorooctanoate as a means of preventing excess oil flow over the output electrodes. We gauge the quality of droplet extraction by measuring the dodecane percentage in our extracted droplet. We explore the effects of coverslip height, coverslip material, coverslip edge geometry, and oleophobic region geometry on the droplet extraction from an electrowetting-on-dielectric microfluidic device.
II2: Clinical Diagnostic Devices
Monday PM, November 28, 2011
Room 206 (Hynes)
2:30 PM - **II2.1
Development of Electrochemiluminescence and Surface Plasmon Resonance-Based Immunosensors with Surface Accumulable Molecules.
Ryoji Kurita 1 Show Abstract
1 , National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
The electrochemiluminescence (ECL) and surface plasmon resonance (SPR) based immunosensors for measuring a trace level of disease markers are shown. It is well known that thiols form a self-assembled monolayer on a metal surface, and this has been widely used to modify metal surfaces. We employed this characteristic for a highly sensitive immunosensors by obtaining a surface pre-concentration of thiol molecules formed by the enzymatic reaction of labeled antibody.Thiocholine, a kind of thiol, was found to be a very useful coreactant with tris(2,2′-bipyridyl)ruthenium complex for bright ECL emission because thiocholine has the bifunctional properties of ECL acceleration and surface accumulation on a gold surface by the gold-thiol binding. Therefore, we propose an ECL based enzyme-linked immunosorbent assay (ELISA) employing a labeling enzyme that produces thiocholine. This marks the first time that ELISA and ECL detection have been combined. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by the gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs. Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of Ru complex. Our new method was successfully employed to realize a high signal to noise ratio immunoassay thus enabling us to measure trace levels of disease marker protein and DNA.We also applied a micro immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable SPR system. Sample BNP solution was introduced into the micro immunosensor after an immuno-reaction with acetylcholine esterase labeled antibody (conjugate) and only unbound conjugate was trapped on the BNP immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multi-step immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device.
3:00 PM - II2.2
Multiplexed, Enzyme-Free Pathogen Detection Using a DNA Nanobarcode Microfluidic Device.
Roanna C. Ruiz 1 , Mark Hartman 2 , Hector Acaron 2 , Thua N. Tran 2 , Shawn Tan 1 , Dan Luo 2 Show Abstract
1 Biomedical Engineering, Cornell University, Ithaca, New York, United States, 2 Biological and Environmental Engineering, Cornell University, Ithaca, New York, United States
Identifying specific pathogens via nucleic acid-based tests is becoming an essential detecting method for a variety of fields including medicine, agriculture, and food safety. Although current assays such as polymerase chain reaction (PCR) have high sensitivity, these methods have certain limitations: they can be time consuming, require specially trained personnel, enzymes, and expensive equipment, and typically only detect one pathogen per test. To overcome these challenges, there is a need for user-friendly point-of-care (POC) devices that perform multiplexed sensing in an automated, efficient, and cost-effective manner. Due to enhanced controllability and diverse design capability, DNA nanotechnology is an ideal approach to interface with nucleic acid materials, offering an attractive alternative to current pathogen detection methods. Towards this end, we have utilized DNA as a structural polymer to design novel branched-DNA-based “fluorescent nanobarcodes” that can identify multiple pathogens simultaneously in a single test using a non-amplified sample. Each DNA nanobarcode carried a unique fluorescent color ratio code that corresponded to a specific pathogen nucleic acid (DNA and/or RNA). Our detection approach was implemented in a microfluidic device that identified pathogens via an addressable array of DNA nanobarcodes. Our microfluidic device provides a novel enzyme-free platform for automated, efficient, and cost-effective multiplexed detection of pathogens.
3:15 PM - II2.3
Label-Free Biomolecule Detection in Nanowall Arrays.
Takao Yasui 1 2 , Noritada Kaji 3 , Yukihiro Okamoto 2 , Manabu Tokeshi 1 2 , Yasuhiro Horiike 4 , Yoshinobu Baba 1 2 5 Show Abstract
1 Applied Chemistry, Nagoya University, Nagoya Japan, 2 FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya Japan, 3 ERATO Higashiyama Live-Holonics Project, Nagoya University, Nagoya Japan, 4 , National Institute for Materials Science, Tsukuba Japan, 5 , National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu Japan
Recently, many researchers have paid attention to lab-on-a-chip (LOC) or micro-total-analysis-systems (µTAS) devices due to an advantage which these devices could integrate through whole laboratory procedures in biotechnology and chemical industry. Despite the fact that the pretreatment system and separation system showed a great progress in nearly a decade, the detection system considerably made a slow progress. During the past decades, numerous nanostructures to separate DNA and protein molecules have been reported, since they have tremendous advantages to realize ultra-fast analysis of biomolecules with ultra small sample consumption, compared with the conventional gel-based separation technologies, including microchip, capillary, or gel electrophoresis. On the other hand, the detection system still relies on the fluorescenct based detection. The reason is that it is difficult to detect the bare biomolecules due to a limitation of microscopic resolution, and therefore, many reports have been forced to use fluorescent molecules to detect separated biomolecules. As for the judgement for whether a particular molecule resides or not, it is no problem to label it with fluorescent molecules, but when it comes to clinical applications, it is of a great importance to utilize the bare biomolecule separated and extracted from samples. The technique to detect biomolecules with no fluorescent molecules has been desired. In this time, we demonstrated a new detection method using nanostructures without any fluorescent molecules. This method we develop uses the diffraction phenomenon which nanostructures have intrinsically. Another benefit using nanostructures is that they have a potential to be a sieving matrix for biomolecules.In this study, an array of nanowall, which is 600 nm wide and 2.7 µm high, was fabricated on a quartz chip by electron beam lithography, photolithography, and plasma etching, as described elsewhere. We precisely controlled the nanowall spacing of 200 nm. We used 532 nm emission line of a diode pumped solid-state laser with output power of 20 mW for the incident laser. A light chopper modulated the incident laser with a modulation frequency at 1 kHz. The laser was focused by 10×/0.30NA objective lens and diffracted by nanowall arrays. The diffracted laser was detected by a photodiode and fed into a lock-in amplifier. The time constant of the lock-in amplifier was set to 100 ms. For the performance assessment of our label-free detection system, we measured the signal shift from air to water, and had a result of S/N ratio over 300. We also detected the difference between water and 3×TBE (267 mM Tris-Borate and 6 mM EDTA) buffer. Finally, we could distinguish the signal from native DNA molecules. Owing to the availability to separate DNA molecules in the nanowall chips, it will be feasible to separate and detect label-free DNA molecules simultaneously with the nanowall chips.
3:30 PM - **II2.4
Plasmonic Metamaterials for Multi-Resonant Spectroscopy and High Resolution Optical Trapping.
Hatice Altug 1 , Arif Cetin 1 , Mustafa Turkmen 1 , Kai Chen 1 , Serap Aksu 1 , Ali Yanik 1 , Alp Artar 1 Show Abstract
1 Electrical and Computer, Boston University, Boston, Massachusetts, United States
Sensitive and quantitative detection of large variety of biological and chemical compounds is needed for early disease diagnostics, environmental monitoring, and homeland security. In this talk, we will introduce plasmonic bio-chemical sensor platforms enabling ultrasensitive label free detection, resonantly enhanced multi-color vibrational spectroscopy as well as high-resolution optical trapping and manipulation. Within the last decades several innovative optical detection technologies have been investigated for sensitive, label-free, quantitative and multiplexed bio-chemical sensing and spectroscopy. Optical biosensors allow transduction of the biomolecular binding signal remotely from the sensing volume, thereby minimizing sample contamination. Unlike mechanical and electrical sensors, they are also compatible with physiological solutions and are not sensitive to changes in the ionic strengths of the analyte solutions. Among optical sensors, methods exploiting surface plasmon resonance (SPR) are taking the lead. Surface plasmons, electromagnetic excitations propagating at metal/dielectric interfaces associated with the collective oscillations of electrons, can interrogate minute changes within the close vicinity of the interface. In fact, SPR is considered as the gold standard technique for label free-detection. Furthermore, plasmons by enabling extreme concentration of electromagnetic fields much below the diffraction limit, strong light-matter interaction and large optical gradients are being explored for highly sensitive surface enhanced spectroscopy as well as for optical trapping and manipulation of analytes. In this talk, we will introduce several plasmonic sensor. First, we will describe a structure which consists of arrays of gold nanopillars for biosensing, nanospectroscopy and optical trapping at the same time. The structure combines the complementary characteristics of localized and extended surface plasmons. We demonstrate high refractive index sensitivities (~675 nm/RIU, where RIU stands for refractive index unit) with large figure of merits (~110). Such good detection limits are attributed to tight localization of plasmonic excitations in nanopillars structures leading to spectrally narrow resonances with enhanced near-field intensities. Plasmonic hot-spots created at the tips of the nanopillar structures also allow trapping of nanoparticles with optical forces as high as 350 pn/W/um^2 at low power excitation sources. We also show that these strong forces around the hotspots can be controlled by incident light polarization allowing direct manipulation of bio-chemical analytes on-chip. Next, we will describe multi-resonant plasmonic metamaterials supporting high optical fields. We will show that these structures can be used to simultaneously enhance different vibrational modes of bio-chemical compounds. Accessing multiple molecular bands is crucial to specifically and accurately identify unknown biochemical.
4:30 PM - **II2.5
Microfluidics for Cell Sorting and Clinical Applications.
Mehmet Toner 1 Show Abstract
1 MassGeneral Hospital, Harvard Medical School, Charlestown, Massachusetts, United States
Bodily fluids, especially blood, contain a treasure of information about the functioning of whole body. Consequently, blood sampling and analysis are of prime interest for both clinical and biomedical research applications, and hold a central place in the diagnosis of many physiologic and pathologic conditions, localized or systemic. However, tapping into this wealth of information has been significantly limited with the lack of adequate technologies and the unspecific nature of the information generated from the current approaches. Among the new technologies with an increasingly broader impact in biology, microfluidics is extremely attractive for blood and other bodily fluid analysis. This presentation will focus on our recent efforts to bring microfluidics to clinical medicine in (i) cancer, (ii) burns and trauma, and (iii) global health. While each of these applications has drastically different design and engineering requirements, the capture of specific cells in peripheral blood is achieved through the use of binding of target cells to antibody-coated surfaces in precisely controlled micro-channel flows. In cancer, the use of microfluidics in isolating extremely rare circulating tumor cells (CTCs) from ~5 to 10 mL of whole blood and the development of CTC-chip will be discussed with specific examples for the initial utility of the CTC-chip in various cancers. In burns and trauma, a point-of-care microchip to isolate homogeneous population of inflammatory cells from ~100 to 300 μl of whole blood will be presented to obtain both genomic and proteomic information from blood cells without altering their biology. In global-health application, label-free isolation of CD4+ T-cells for monitoring HIV/AIDS patients in highly resource-limited environments from a fingerprick of whole blood (~5 μl) will be discussed. In all these clinically relevant examples, the importance of using samples from real patients in the development of new technology will be emphasized. The challenges and perils in bridging technology development with clinical medicine will also be discussed in the context of these three distinct applications.
5:00 PM - **II2.6
Immuno-Pillar Chips for Clinical Diagnosis.
Manabu Tokeshi 1 2 Show Abstract
1 Department of Applied Chemistry, Nagoya University, Nagoya Japan, 2 FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Nagoya Japan
Recently, a concept of "plasma proteome profiling", which a lot of plasma proteins are measured at the same time, has paid much attention to clinical diagnostics [1,2]. By using data from the plasma proteome profiling, we are expecting that a highly accurate diagnosis becomes possible. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are currently used in routine clinical diagnostics. One reason for this is that there is no method of measuring a lot of plasma proteins with greatly different concentration range easily and rapidly. Very recently, Heath et al., developed a new device (an integrated blood barcode chip) that can detect a dozen different proteins in whole blood simultaneously , although this cannot detect wide dynamic range of proteins. These approaches will bring us big benefits: improvement of diagnostic accuracy, early detection of disease, proper treatment depends upon the stage of the disease, and so forth.We are advancing the research aiming at the development of a plasma profiling device for clinical diagnostics. To realize the device, it is necessary to satisfy all the requirement for the device: i.e., provide rapid analysis with high sensitivity, have wide dynamic range, be easy-to-use, require small volumes of sample and reagents, and be fabricated at low cost. Very recently, we developed a new device called an "immuno-pillar chip ", which has the desired features for a plasma profiling device. It has hydrogel pillars (diameter: 200 µm, height: 50 µm) fabricated inside a microchannel, with many antibody molecules immobilized onto 1 µm diameter polystyrene beads. For detection of disease markers, we confirmed the chip provides rapid analysis (total assay time: 4-12 min) with high sensitivity, it is easy-to-use (no special skills are needed), and it uses small volumes of the sample and reagent (0.25 µL each for plasma or 2 µL each for whole blood). Moreover, multiplex assay for three biomarkers was also possible.We are working on the development of a multi-biomarker detection chip for the diagnosis of diabetic nephropathy by using this device in cooperation with the Department of Medicine at Nagoya University now. We believe that the plasma profiling chip for clinical diagnostics can be developed by expanding the function of the immuno-pillar chip.N. L. Anderson, N. G. Anderson, Mol. Cell. Proteomics, 1, 845–867 (2002).P. Mitchell, Nat. Biotechnol., 28, 665 (2010).R. Fan, O. Vermesh, A. Srivastava, B. K. H. Yen, L. Qin, H. Ahmad, G. A. Kwong, C. -C. Liu, J. Gould, L. Hood, J. R. Heath, Nat. Biotechnol., 26, 1373-1378 (2008).M. Ikami, A. Kawakami, M. Kakuta, Y. Okamoto, N. Kaji, M. Tokeshi, Y. Baba, Lab Chip, 10, 3335 (2010).
5:30 PM - II2.7
On-Chip Diagnostic System for Circulating Tumor Cells.
Jaehoon Chung 1 , Huilin Shao 1 , Ralph Weissleder 1 , Hakho Lee 1 Show Abstract
1 Center for Systems Biology, Massachusetts General Hospitals, Boston, Massachusetts, United States
We have developed a novel, low-cost and high-throughput microfluidic device for detection and molecular analysis of circulating tumore cells (CTCs). The device captures CTCs directly from unprocessed whole blood, provides on-chip cell labeling for CTC identification, and allows facile cell-retrieval for further analyses. The device operation is based upon a size-selective cell separation technique, which was implemented by a weir-style physical barrier with a gap in the main fluidic channel; blood cells which are smaller than the gap height move straight through following a laminar flow, whereas larger cancer cells deviate from their original path and move along the physical barrier to be collected in a separate outlet. This new system is a versatile CTC analysis platform with many advantages. First, it supports extremely high throughput operation, since the use of weir structure effectively reduces fluidic resistance and enables flow-through separation.Specifically, we achieved >6000-fold CTC enrichment from whole blood at a high flow rate (10 mL/h). Second, the CTC-chip facilitates clear visual verification and enumeration of captured cells during/after operation. For this purpose, we have implemented microwell-shaped capturing structures on the physical barrier. Cancer cells introduced to the device were individually collected at each capture site, allowing single-cell resolution analyses. Furthermore, the captured cells could be profiled in situ by introducing fluorescent antibodies. The chip thus assumes not only high detection sensitivity but also molecular specificity for CTC identification. Finally, the CTC-chip provides a facile way to retrieve captured CTCs. By reversing the flow direction, the cells can be dislodged from their capture sites and collected for downstream investigation (e.g., cell culture and genetic analyses).
5:45 PM - II2.8
Cancer Cells in 3D Microenvironments: Individual and Collective Migration Behaviors.
Ian Wong 1 2 , Daniel Irimia 1 2 , Mehmet Toner 1 2 Show Abstract
1 BioMEMS Resource Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States, 2 , Harvard Medical School, Boston, Massachusetts, United States
The metastasis of cancer cells from the primary tumor to surrounding tissues in the body is ultimately responsible for 90% of cancer-related deaths. However, the mechanistic details of how malignant cells invade into the tissues and vasculature are poorly understood. Here, we use microfabricated 3D environments with controlled chemoattractant gradients to directly characterize individual and collective migration of cancer cell populations with controlled degrees of malignancy. Using automated image analysis techniques, the behavior of these heterogeneous subpopulations can be distinguished and categorized. Finally, this platform is implemented as a high-throughput screen to quantitatively measure the effects of small molecule anti-metastatic therapies.
II3: Poster Session
Monday PM, November 28, 2011
Exhibition Hall C (Hynes)
9:00 PM - II3.1
Evaluation of Performances of Organic BioMEMS as Resonators.
Georges Dubourg 1 , Isabelle Dufour 1 , Claude Pellet 1 , Cédric Ayela 1 Show Abstract
1 , IMS laboratory, Talence France
Polymers are promising materials for MEMS and sensor applications. They are particularly attractive for sensitive biosensing applications due to their low cost, good processability, bio-compatibility and tuning properties, such as Young’s modulus. Actually, an organic free-standing structure is more flexible than a silicon one: the use of an organic microcantilever offers the possibility of characterizing many deformations in dynamic mode that are difficult or impossible to be observed with silicon structures. Thereby, an organic cantilever can operate at high resonant frequencies improving their sensitivity for sensing applications. In the same time, the common fabrication methods are mainly restricted to photosensitive materials. Therefore, the introduction of versatile methods to pattern materials such as thermoplastics and biopolymers which are not affected by the standard photolithography is challenging. In this context, the presented work proposes a new collective microfabrication process of all-organic microcantilever chips made of PMMA material. This method is based on the hierarchical combination of shadow-masking and wafer-bonding processes. The shadow-masking combines deposition and patterning in one step thanks to spray-coating through a polymer microstencil that gives the opportunity of patterning thermo and photo sensitive materials. The resulting organic structures are then transferred onto SU-8 chips by using an SU-8 wafer-bonding process that is well-adapted for the wafer-level fabrication of organic cantilevers.The second part of this study focuses on the dynamical characterization of resulting microstructures as resonators to evaluate the structured material performances. The polymer-based microcantilevers are actuated by the electromagnetic Lorentz force obtained by an external magnet and an alternative electrical current in a conducting path integrated on the structure. For this, the gold path is deposited on the structures by thermal evaporation through polymeric microstencil. With this approach, fifteen out-of-plane resonant modes have been measured, including torsional and flexural ones. Resonant frequencies above 1MHz have been obtained for the fifteenth mode enhancing the organic structure sensitivity of 2 decades compared to the first mode. Thereby, organic cantilevers used as resonator have the potential to serve as highly sensitive devices for biological sensing applications. In addition, the unconventional use of microcantilevers in dynamic mode knows a fast-growing interest. More specifically, the use of cantilevers in the in-plane vibration modes may be of potential interest for detection in liquid media where viscous damping occurs. Currently, a piezoelectric material is mandatory to generate this mode while, in our case, the flexibility of organic cantilevers will allow the direct observation of the first longitudinal resonant mode.
9:00 PM - II3.10
Functional Design of Porous Drug Delivery Systems Based on Laser Assisted Manufactured Nitinol.
Igor Shishkovsky 1 Show Abstract
1 Laboratory of Technological lasers, Lebedev Physics Institute of Russian Academy of Sciences, Samara branch, Samara Russian Federation
Previous our studies have shown the presence of shape memory effect (SME) in the biocompatible porous nitinol (intermetallic phase NiTi), fabricated by the selective laser sintering (SLS) method . In the living tissue under raise of temperature (beginning disease) the size of pore will decrease an account of austenite phase transformation and a pharmaceutical composition will extrude from the pores. And wise versa on the cooling stage (a tissue temperature returns to normal) the intake of drug will stop. Depending on the type of the three dimensional structure of scaffold, determined on the stage of computer aid design, the velocity of penetration is possible to control. Because the scaffolds consist of random pores, we propose to derive the inhomogeneous surface strain distribution numerically by combining micro-compression experiments with Finite Element (FE) model. . Shishkovsky I.V. Laser synthesis of functional mesostructures and 3D parts. Moscow. Fizmatlit Publ.: 2009. ISBN 978-5-9221-1122-5. 424 p.
9:00 PM - II3.11
Study of Double Emulsion Behavior by Optical Force.
Kyungheon Lee 1 , Sang Bok Kim 1 , Kang Soo Lee 1 , Seung Hwan Kim 1 , Sang Youl Yoon 1 , Hyung Jin Sung 1 Show Abstract
1 Mechanical Engineering, KAIST, Daejeon Korea (the Republic of)
Optical force has been used in numerous fields as tool to manipulate micro particulate, such as cell, micro/nano particles and bio-molecules. Because of its non-invasive nature, many research fields adapted optical force for manipulating single or multiple micro particulate. Among several micro particulates, single and multiple layered emulsions are used in many research fields. Encapsulation of specific ingredient with emulsion is widely used to chemical science, biological cell encapsulation study, drug delivery, food and synthesis of specific shape of micro particulate. Several parameters determine the size of each emulsion and its frequency such as flow rate of each fluid, geometry of generating device and fluid properties. In order to obtain high quality encapsulation and other synthesis, it is very crucial to manipulate emulsions based on its size and ingredient properties.In the present work, we demonstrate manipulation of double emulsion that depends on differences of refractive index and relative size between inner and outer emulsion by optical force. We derived and calculated the analytic expression of optical force on a pair of concentric spheres with photon stream method and measured the behavior of double emulsion by the optical force. The behavior of double emulsion was also calculated with the optical force distribution and particle motion governing equation. For the experimental measurement, a simple device for generating double emulsion with surface treated co-flowing geometry was employed and laser beam propagate perpendicular to the double emulsion and outer fluid flowing direction. When the double emulsion passed through the laser beam, the scattering force pushed them in the direction of laser beam propagation and the double emulsion move their position in the plane perpendicular to the fluid flow. The shifted distance was controlled by emulsion size ratio, refractive index and other optical parameters of double emulsion. To identify the refractive index of selected fluid mixture, defocusing based micro refractometer measurement was carried and other parameters were also controlled. The analytical results were compared with experimental data and were found in good agreement. This work has potential uses in double emulsion sensing, separation for drug delivery and emulsion related biomedical applications.
9:00 PM - II3.12
Planar Impedance Sensing Device for Cellular Response Studies.
Jinwang Tan 1 , Xin Zhang 1 Show Abstract
1 , boston university, Boston, Massachusetts, United States
This project is aiming to develop a cell impedance sensing and analysis system for cellular response studies. Cellular responses are kept close eye on in the experiments with cell models where a variety of labeling techniques and optical observation are the most common approaches. Despite the impressive achievement attained with these methods, current and future studies strive to provide effective and quantitative detection, capable of achieving real-time monitoring of transient cell responses. Bioelectronics techniques, with the help of advancement in automatic detection, have gradually been utilized by biomedical researchers to study the cellular responses. These techniques, such as electrical cell-substrate impedance sensing (ECIS) and real-time cell electronic sensing (RT-CES), have been developed to determine the cell-substrate or cell-cell adhesion based on the average morphology of a large number of cells. The advancement of shrinking electrode dimension into subcellular level will provide even more undisturbed cell morphology and electrical properties with isolated cells.The planar electrodes have been created with microfabrication processes to carry out the impedance measurement of certain electrode/electrolyte interface.As the first step of this project, we have developed our device for cell sample preparation and chemical delivery.The device consists of the following functional sections: 1) A microfluidic section that delivers cell suspension and chemicals with well designed network; 2) An alternating current electrokinetics (ac-EK) section that positions HEK cells onto the sensing electrodes by dielectrophoresis. The novel design of the electrode array makes it serve as both trapping and sensing electrodes, which significantly reduce the complexity of our design as well as the cost of fabrication. Following steps such as cyclic spectrum measurements of working electrode impedance and automatically data fitting for electrochemical impedance spectroscopy (EIS) will be performed in the future.
9:00 PM - II3.13
Nanostructured Selenium for Preventing Biofilm Formation on Medical Devices.
Thomas Webster 2 , Qi Wang 1 Show Abstract
2 School of Engineering, Brown University, Providence, Rhode Island, United States, 1 Department of Chemistry, Brown University, Providence, Rhode Island, United States
In this study, we coated traditional implants with selenium nanoparticles to impart antibacterial properties directly onto the surface of medical devices. Selenium can kill bacteria by depleting their thiol levels. The nano-scale size of selenium nanoparticles increases the surface area of selenium available to interact with and kill bacteria. For this, selenium nanoparticles were synthesized through a simple reaction between glutathione and sodium selenite (4:1 molar mixture) and at the same time were coated on the surface of various medical devices. These substrates included PVC (polyvinyl chloride), polycarbonate, PU (polyurethane), co-polyesters and silicone. After coating, tape tests and fluid flow assays were used to test the strength of adhesion of the selenium nanoparticles on the substrate surfaces. SEM images of the substrate surfaces were taken before and after the adhesion tests to determine coating strength. We achieved very strong adhesion for some of the substrates, like PVC. We also used many methods, such as plasma treatment, UV light treatment, changing temperature, altering pH and coating time, to optimize the coverage and attachment strength of selenium nanoparticles onto all the substrates. Lastly, experiments with bacteria (specifically, Staphylococcus aureus) were conducted to determine the effectiveness of the selenium coating for killing bacteria or preventing bacteria from attaching. Bacteria colonization decreased significantly when polymers were treated with selenium coated samples and substrates with higher concentrations of selenium attached less bacteria, which indicated that the selenium coating could inhibit bacteria growth and deserves further investigation.
9:00 PM - II3.14
Facile Technique for Cell Patterning and Multiple Cell Types Co-Culturing.
Alexander Efremov 1 2 , Eliana Stanganello 1 , Steffen Scholpp 1 , Pavel Levkin 1 2 Show Abstract
1 Department of Toxicology, Karlsruhe Institute of Technology, Karlsruhe, Baden-Württemberg, Germany, 2 Department of Applied Physical Chemistry, University of Heidelberg, Heidelberg, Baden-Württemberg, Germany
The ability to control spatial arrangement of different cell types is crucial for in vitro cell function studies, for designing of tissue constructs that mimic the organization of in vivo cell compartmentalization and variety bioassays . Although the existing cell patterning technologies allow co-culturing of different cell types, they are usually limited to relatively simple geometries. On the contrary, methods used for obtaining complex geometries are usually applicable for patterning of only one cell type. We have developed a facile method enabling conjoint culturing of more than one cell types in areas with virtually unlimited geometrical complexity.Our method is based on the formation of highly hydrophilic (HH) areas (17.4°±0.5°, static water contact angle) surrounded by superhydrophobic (SH) borders (146.1°±2.3°, advancing water contact angle). The HH/SH patterned surface is fabricated by first formation of a nanoporous HH poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) polymer layer on a glass plate followed by UV-initiated fluorination of its surface through a photomask. The surface modification method we use is based on grafting of poly(2,2,3,3,3-pentafluoropropyl methacrylate) brushes on the surface of HEMA-EDMA matrix using photografting. The very high difference in wettability of hydrophilic areas and the surrounding SH border allows us to enclose aqueous solutions (e.g. cell suspensions) inside the hydrophilic areas. Thus, the cell patterning is carried out by filling separated hydrophilic reservoirs with suspensions of different cell lines followed by the co-culturing of adhered cells in the same medium.We characterized the patterned surfaces by scanning electron microscopy, X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. The nanoporous structure of the polymer film assures transparency of sample that permits monitoring of cells by means of optical microscopy. HH/SH surfaces with different geometries of patterned area were utilized to control spatial arrangement of different cell lines on the substrates. We also performed patterning of primary embryonic zebrafish cells to mimic formation of Sonic Hedgehog gradients in vitro. Our preliminary results confirmed good adhesiveness and viability of the tested cell lines. The smallest obtained distance between hydrophilic reservoirs divided by a SH gap was 30 μm that corresponded to the average diameter of a eukaryotic adhered cell. Close proximity of patterned areas, flexibility in pattern geometry, transparency of the polymer film, good cell adhesiveness and viability as well as versatility of the method for the preparation of patterned substrates make the technique an excellent approach for cell patterning, mimicking natural cell arrangements in vivo, studying cell-cell communications and application in a variety of bioassays. Kaji et al. Biochimica et Biophysica Acta, 2011 Zahner et al. Adv. Mater., 2011
9:00 PM - II3.17
Metal Assisted Plasma Etching (MAPE).
Teena James 1 , David Gracias 1 Show Abstract
1 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States
We describe a novel phenomenon of metal assisted plasma etching of silicon (Si) substrates. Etching is accelerated in the vicinity of noble metal patterns resulting in the single-step formation of novel structures for biomedical applications. These structures include nanoparticle coated microfluidic channels that have been used for sensing. We also describe the creation of nanoporous silicon membranes (with conical pores) and describe their applications in biomolecular separations and ion rectification.
9:00 PM - II3.18
Development of a Microscale Uni-Axial Loading Device for Intercellular Mechanotransduction Study.
Qian Wang 1 , Yi Zhao 1 Show Abstract
1 Biomedical Engineering Department, Ohio State University, Columbus, Ohio, United States
This paper reports development of a microdevice that can deliver controllable uni-axial stress to live cells, where controllable tensile or compressive stress can be loaded on selected cells while keeping other cells unloaded. The propagation of mechanical signals regulated by cell-cell communication can thus be quantitatively studied.Cells in tissues in vivo are constantly subjected to mechanical loads. These mechanical signals are essential for maintaining cellular functions. Current loading devices often expose all cells in culture to one loading condition at the same time. Mechanical loading to selected cells is yet to come. In this work, a device that can apply controllable compressive/tensile uni-axial loads to selected cells is demonstrated. The device consists of two polydimethylsiloxane (PDMS) substrates. The top substrate consists of an array of rectangular membranes. Each membrane is 7500μm in length, 500μm in width, and 50μm in thickness. The bottom substrate contains a microfluidic network. Upon loading, the fluid in the microfluidic channel deforms the membrane and applies stress to cells cultured on the top surface of the membrane. Given the large length-to-width ratio, uni-axial loads can be applied. In this design, each membrane is independently deformable to deliver desired levels of strain to cells in different regions.To examine the actual strain, a microdots array (5μm in diameter and single spaced) is patterned on the membrane of the top substrate. Displacements of the dots upon membrane deformation are optically determined, where the three-dimensional profile of the membrane can be reconstructed. In–plane strain field is then derived from the displacement map of the microdots array using classic large strain/displacement equations with the Lagrangian strain operator. The result shows that the uni-axial strain at the membrane center ranges from about 5% compressive to about 25% tensile, validating the capacity of the device in applying both tensile and compressive loads. A wider range can be achieved by adjusting the membrane geometries and the pumping parameters.Cell testing is performed using murine skeletal myoblast cell line C2C12. After culturing C2C12s in the device and exposing them to 0.5Hz cyclic strain (-5% to 10%) for 4 days, the cells align along the length direction. The myoblasts are then allowed to differentiate into myotubes. The result shows that not only the myotubes on the loading membranes exhibit linear alignment, but also the unloaded myotubes between the loading membranes align in the same direction. The alignment efficacy is dependent on the distance between the loading sites. Such effect is believed due to cell-cell communication. This work validates that the reported microdevice is capable of investigating cell-cell communication by selectively loading certain cells in culture while leaving other unloaded, which holds a promise for intercellular mechanotransduction studies.
9:00 PM - II3.19
Adhesion and Cohesion in Structures Containing Suspended Microscopic Polymeric Films.
Wanliang Shan 1 2 3 , Jing Du 1 2 , Emily Hampp 1 2 , Hannah Li 3 , George Papandreou 3 , Cynthia Maryanoff 3 , Wole Soboyejo 1 2 Show Abstract
1 Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey, United States, 2 , Princeton Institute for the Science and Technology of Materials, Princeton, New Jersey, United States, 3 , Cordis Corporation, a Johnson and Johnson Compay, Spring House, Pennsylvania, United States
This paper presents a novel technique for the characterization of adhesion and cohesion in suspended micro-scale polymeric films, which is based on a combination of experiments and computational models. On such films, load is applied using probes that were fabricated by focused ion beam (FIB) techniques. The underlying stresses associated with the different probe tip sizes were computed using a finite element model (FEM). The critical force for failure of the film substrate interface is used to evaluate adhesion, while the critical force for the penetration of the film evaluates cohesion. When testing a standard material, polycarbonate, a shear strength of approximately 70 MPa was calculated using Mohr and Coulomb’s theory; this value is in agreement with literature results. The technique was applied to the measurement of adhesion and cohesion in a model drug-eluting stent called NEVOTM Sirolimus Eluting Coronary Stent (SES), which contains suspended polymeric films in metallic Co-Cr alloy reservoirs. The cohesive strength of the formulation was found to be comparable to that of plastics.
9:00 PM - II3.20
Visualization of NIR Propagation in Quasi-Zero Index Photonic Crystals Using Upconverting Nanoparticles.
Jingyu Zhang 1 , Daniel Gargas 1 , Teresa Pick 1 , Scott Dhuey 1 , Emory Chan 1 , Alexis Ostrowski 1 , Brett Helms 1 , James Schuck 1 , Deirdre Olynick 1 , Stefano Cabrini 1 Show Abstract
1 , Lawrence Berkeley National Lab, Berkeley, California, United States
Lanthanide-doped upconverting nanoparticles have interesting properties for bioimaging. Here, we present the use of upconversion nanoparticles (NaYF4: Er3+) to image near infrared (NIR) light propagation in photonic crystal (PC) waveguide with conventional optical microscopy. The PC structure is composed of a subwavelength negative index (n=-1) PC slab and a positive index (n=1) air slab in periodic arrays, which show quasi-zero refractive index (QZRI) and collimate 1.55µm wavelength light propagation beyond the diffraction limitation over millimeters. Er3+ doped NaYF4 nanoparticles were used to convert 1.55 µm to visible light through a multi-photon absorption based on sequential energy transfers involving real metastable-excited states. Nanoparticle-assisted NIR light mapping has distinct advantages over other methods such as typical NIR setups which are limited by the NIR wavelength, or the expensive near field scanning optical microscope (NSOM) which is limited by extremely shallow depth of field and long scanning times. In our technique, nanoparticles on PC waveguides are illuminated by a continuous wave laser to generate upconverted luminescence with high sensitivity to local field intensity at optical wavelength resolution. The QZRI PCs on SOI wafer were prepared by electron-beam lithography and cryo-Si plasma etching. We optimized the synthesis, surface treatment, concentration, and deposition methods of the nanoparticles solution in order to produce a layer of 10-20 nm Er3+ doped NaYF4 nanoparticles uniformly distributed on the surface of the PC holes over a large area (2 x 2 mm). Such a layer does not significantly change the PC refractive index. On such structures, the photoluminescence (PL) intensity (530/550 nm) dependence of pumping intensity (1550 nm) has been measured and used to quantify the enhanced local field intensity confined in photonic crystals.
9:00 PM - II3.4
Apatite Coating on Porous Silicone for BioMEMS.
Luci Cristina Vercik 1 , Thiago Antonio Menezes 1 , Leticia Baptista 1 , Andres Vercik 1 Show Abstract
1 Basic Sciences Department - FZEA, Universidade de Sao Paulo, Pirassununga - SP Brazil
The Bio-Electro-Mechanical Systems are built using the well-known fabrication processes of microelectronic industry and incorporate a biological component into its structure. These devices are widely used as biosensors and different kinds of actuators with applications in several areas of technology such as pharmaceutical and food industry, biomedical and environmental monitoring. Lab-on-a-chip systems and micro-total analysis systems (micro-TAS) using cantilever structures are used for detection of DNA hybridization and could be used to detect virus, proteins, microorganisms and small molecules of biotechnological interest. Despite the biocompatibility of BioMEMS, when used as an implantable device, is mainly concerned with the preservation of the tissue function, the lack of this property can also affect de device performance and functionality. To overcome this drawback, several surface modifications have been proposed to enhance the biocompatibility and to avoid biofouling. An alternative is coating the surface with a biocompatible material such as an apatite layer. In this work the coating of porous silicon (PSi) with hydroxyapatite was addressed. The PSi is a material often used in MEMS fabrication and has excellent optical properties, which make it suitable for optical biosensors. Porous silicon was obteined by electrochemical etching of crystalline <100> p-type silicon of microelectronic quality with resistivity between 4–40 Ωcm, wafer thickness of 525μm, previously cleaned with RCA standard processes, using an electrolyte of HF:Ethanol:H2O (1:1:5) and a current of 10mA for 10 minutes. After the porosification of silicon, the samples were treated with 0.5mol/L NaOH solutions for 120 minutes and then immersed in a Simulated Body Fluid (SBF) solution at 37°C for 7 days whereas other samples were coated without NaOH treatment. MEV, FTIR and DRX allowed observing that the samples without pretreatment in NaOH solution were not coated, as expected, whereas the pretreated sample exhibited a uniform apatite coating. FTIR showed absorption bands near 1415 and 1465 cm-1 attributed to vibrations of the CO32- group. This bands overlapped with the P-O(H) vibration of the HPO42- group, which is characteristic of the octacalcium phosphate. The characteristic band of the A-type substitution was observed at 1547cm-1 as well as the band due to the stretching vibrational mode of the PO43- group. These results indicate the formation of a carbonated hydroxyapatite on the PSi surface.
9:00 PM - II3.6
Engineering Hydrid Cunductive Polymer Microelectrode for Improving Biotic/Abiotic Interface.
Takeo Miyake 1 2 , Yuichi Ido 1 , Daisuke Takahashi 1 , Syuhei Yoshino 1 , Kuniaki Nagamine 1 2 , Matsuhiko Nishizawa 1 2 Show Abstract
1 , tohoku.univ, Sendai Japan, 2 , CREST, Tokyo Japan
Conducting polymers such as poly (3,4-ethylendioxythiophene) (PEDOT) and polypyrrole (PPy) are attractive electrode materials, having the advantages of biocompatibility, high capacitance, and flexibility. They have been utilized in biomedical devices, including implanted electronics and in-vitro devices for culturing cells. We report herein the micropatterning of PEDOT on a hydrogel, such as agarose and collagen, to provide a fully-organic, moist, and flexible electrode [1, 2]. The PEDOT/hydrogel electrodes are prepared through two electrochemical processes: the electropolymerization of PEDOT into the hydrogel and the electrochemical actuation-assisted peeling. The method is versatile and can be used to make micropatterns of PEDOT on other or curvilinear hydrogels.We then demonstrated that the PEDOT/agarose electrode could be used for electrical stimulation of the contractile fibers that make up muscle tissue (myotubes). A film of contractile myotubes within a fibrin matrix was laid on top of the electrode and stimulated with periodic voltage pulses. The electrode induced contraction of the myotubes, and the electrode itself was observed to contract in unison with the myotubes. Sekine, S.; Ido, Y.; Miyake, T.; Nagamine, K.; Nishizawa, M., J. Am. Chem. Soc. 2010, 132, 13174–13175. Nature Asia Materials, doi:10.1038/asiamat.2010.173
9:00 PM - II3.7
Complex Modulus Study of PDMS by Dynamic Nanoindentation.
Ping Du 1 , Chen Cheng 2 , Hongbing Lu 2 , Xin Zhang 1 Show Abstract
1 Mechanical Engineering, Boston University, Boston, Massachusetts, United States, 2 Mechanical Engineering, University of Texas at Dallas, Richardson, Texas, United States
A key issue in using Polydimethylsiloxane (PDMS) based micropillars as cellular force transducers is obtaining an accurate characterization of mechanical properties. The Young’s modulus of PDMS has been extended from the ideal elastic constant to the time-dependent viscoelastic function in our previous work. However, the frequency domain information is of more practical interest in interpreting the complex cell contraction behavior. In this work, we investigated the complex modulus of PDMS by using the dynamic nanoindentation technique (DNT). The effects of curing condition and storage time on the modulus were evaluated. The PDMS samples were in a thin-film format with a thickness of ~3 mm. The samples were prepared by mixing the prepolymer Sylgard 184 (Dow Corning) with a curing agent at a volume ratio of 10:1, degassing and then thermal curing in oven. One of the major factors which will affect the mechanical properties of the PDMS samples is the curing condition. Therefore in this work we adopted two schemes: 65 °C for 90 min and 80 °C for 120 min. For the first scheme, three samples prepared and stored for different times were selected to study the effect of storage time on the mechanical properties. The complex modulus was obtained by using the DNT. The DNT tests were conducted by a G200 Nanoindenter system (Agilent) with a sapphire flat-ended cylindrical punch tip (Micro Star Tech.) with a diameter of 2.01 mm. The indenter tip was pre-compressed into the film by a certain depth to assure a full contact, and then a sinusoidal displacement was applied with amplitude of ~50 nm. The indenter tip was vibrated at a discrete number of frequencies in the range of 1-45 Hz, and the corresponding load was measured. After that the complex modulus and loss factor as functions of frequency were obtained from the nanoindenter software package.Both the storage modulus (E’) and loss modulus (E”) clearly show the frequency-dependent behavior: they generally increase as the frequency increases. For the first three samples with the same curing condition, the moduli increase with longer storage times. However, for the sample 4 with higher curing temperature, longer curing time but the least storage time, the moduli are much higher than sample 1 with the longest storage time. Therefore the curing condition has a much profound effect on the mechanical properties of PDMS than the storage time.In summary, we measured the complex modulus of PDMS by DNT, and investigated the effects of curing condition and storage time. We believe this work will help evaluating of the cellular force calculation with more information in the frequency domain.
9:00 PM - II3.8
Solvent-Less Planar Lipid Bilayers Formed in Microfabricated Silicon Chips.
Azusa Oshima 1 , Ayumi Hirano-Iwata 1 2 , Tomohiro Nasu 1 , Yasuo Kimura 1 , Michio Niwano 1 Show Abstract
1 , Tohoku University, Sendai Japan, 2 , Japan Science and Technology Agency (JST), Saitama Japan
Artificial planar bilayer lipid membranes (BLMs) have been used for electrophysiological studies of ion channel proteins. By incorporating ion channels into the BLMs, functional properties of the channel proteins can be analyzed under chemically controlled conditions. However, BLMs prepared by the conventional methods (painting, monolayer folding and tip-dip methods) suffered from instability and lack of reproducibility. Although a variety of approaches for the formation of BLMs in microfabricated apertures and microfluidic channels have been reported, most of the devices were combined with the painting method, leading to the formation of BLMs containing organic solvent. Since organic solvent is likely to denature proteins, there is still a great demand for microfabrication-based methods for preparation of BLMs containing less or no amount of organic solvent. In the present study, we propose a new method for preparation of solvent-less BLMs in a microfablicated silicon chip using monolayer folding method. The basis of the device was a silicon wafer covered with a silicon nitride layer. Microapertures were fabricated in the silicon nitride layer using photolithographic patterning and wet chemical etching. Some of the device was further coated with thermal oxide and Teflon-AF. After treated with a silane-coupling reagent to make the surface hydrophobic, the silicon chip was placed vertically in a Teflon chamber. Artificial BLMs were prepared in the microaperture by folding up two phospholipid monolayers spread at the air/water interface. The BLMs prepared in the microapertures showed the resistance of several tens Gohm and the current noise level was 1-2 pA in peak-to-peak after low-pass filtered at 1 kHz. The BLMs were resistant to applied voltage of ±1 V and the lifetime of the membranes was 15-43 h with and without incorporated gramicidin channels. BLMs containing gramicidin channel were tolerant to repetitive solution exchanges. Such mechanically stable BLMs will open up a variety of applications including high-throughput analysis of ion-channel proteins.
Mehmet R. Dokmeci Harvard Medical School
Brigham and Women's Hospital
Junji Fukuda University of Tsukuba
Ali Khademhosseini Harvard-MIT Division of Health Sciences and Technology
Hirokazu Kaji Tohoku University
II4: Microfluidics for Cellular Microenvironments II
Tuesday AM, November 29, 2011
Room 206 (Hynes)
9:30 AM - **II4.1
Computer Assisted Designing and Biofabrication of 3D Hydrogel Structures towards Thick 3D Tissue Engineering.
Makoto Nakamura 1 , Ken-ichi Arai 1 , Hideki Toda 1 , Shintaroh Iwanaga 1 , Kozo Ito 1 , Genci Capi 1 , Toshio Nikaido 2 Show Abstract
1 Graduate school of Science and Engineering for research, University of Toyama, Toyama, Toyama, Japan, 2 Graduate school of medicine and pharmaceutical science for research, university of toyama, toyama Japan
To break several present limitations in tissue engineering, we have addressed to develop an innovative approach. Biofabrication is defined as the fabricationtechnology focused to produce biological products using living cells and/or biological materials. In the present tissue engineering, engineering of thick functional tissues has been one of the big issues for a long time. To realize to produce such tissues, we need the technologies to fabricate thick and complicated 3D structures composed of multi-cell types. Then, we have developed a custom-made 3D bioprinter using inkjet technology and have achieved to construct several 3D structures directly with hydrogel and living cells together.In this paper, firstly, the recent developments in our 3D bioprinter are reported. We added to our 3D bioprinter an additional printing mode where image based 3D laminating printing is possible. We also added active Z-axis control mode, too. Using the versionupped 3D bioprinter, more complicated structures than before were tried. As a result, we recognized the fabrication performance of the new version printer was significantly developed both in the fabrication of complicated structures and in the fabrication of arbitrary designed structures. We reconfirmed the feasibility of computer-assisted designing and direct 3D fabrication for tissue engineering.Owing to such developments, a next issue has been emerged, that is “What 3D structures should be designed and fabricated for effective incubation?”, because such fabricated bio-products must be kept incubated to advance the following process to develop to physiological bio-structures. Here, we report our recent challenges andachievements. Based on our previous experiments, we recognized that cells can survive only within 100micrometers areas from the surface of the structure by usual procedure of cell culture where oxygen and nutrients can be delivered by passive diffusion. Then, we designed the thick 3D structures with significant perfusion routes. At first, multiple bitmap images of the sequential 2D sections of 3D products are imagined and designed in computer. Next, 3D structures in computer, 3D structures were printed out by our 3D Bioprinter using sodium alginate solution as an ink.As a result, such designed 3D structures could be fabricated successfully. As our 3D Bioprinter can fabricate 3D hydrogel structures also together with living cells, we confirmed the promising feasibility of direct 3D fabrication with living cells. And as those designs were all human arbitrary designs, this results also indicated the possibility of creation of the artificial tissues or artificial bio-devices systems, too. This approach of computer-assisted biofabrication will contribute to further innovative advancement of tissue engineering.
10:00 AM - II4.2
Microfluidic Production of Micro-Assemblies with Multiple Geometries and Functionalities.
Kunqiang Jiang 1 , Don DeVoe 2 , Srinivasa Raghavan 3 1 Show Abstract
1 Department of Chemistry and Biochemistry, University of Maryland-College Park, College Park, Maryland, United States, 2 Department of Mechanical Engineering, University of Maryland, College Park, Maryland, United States, 3 Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland, United States
The concept of “bottom-up” micro-assembly involves precise positioning and robust connecting of individual microsized subunits into more complex, higher-order structures with desired geometries and properties. Specifically, several issues need to be considered in advance before constructing micro-assemblies, including finding suitable subunits, choosing appropriate linking methods, and developing workable spatial templates. It is notable that microfluidics offers an excellent solution to address all of these various issues. Monodisperse subunits can be generated through unique droplet production mechanism, and microfluidic channels can be used as spatial templates to anchor and assemble subunits into complex patterns. Moreover, robust and stable intraparticle linkage can be achieved by adding external physical impulses or by inducing additional chemical crosslinkers, while desired functionalities can be realized by incorporating various encapsulants into the dispersed phase.As a proof-of -concept demonstration, we have successfully utilized microfluidics to produce uniform chitosan microcapsules as subunits (building blocks), and assembled them into various microstructures with facile control over their geometries and properties. The building blocks are microcapsules of the biopolymer chitosan, which are created by dispersing an aqueous solution of chitosan at a microfluidic T-junction with another stream of an immiscible oil phase. In the process, monodisperse chitosan droplets are continuously generated, and these are subsequently crosslinked by a downstream solution of glutaraldehyde (GA). The functional properties of these chitosan capsule properties can be easily varied by introducing various payloads into the disperse phase, such as magnetic nanoparticles and/or fluorescent dyes. We then demonstrate the assembly and linkage of individual capsules into complex structures, again using GA as the chemical “glue”. We have first created linear microchains with tunable flexibility by adjusting the crosslinking conditions: in the case of magnetic chains, both rigid chains that can be rotated by an external magnetic field as well as semiflexible chains that show a beating motion have been produced. The arrangement of capsules within a chain can also be precisely controlled, e.g., to generate linear chains with alternating fluorescent and non-fluorescent capsules. Besides, other complex structures can also be created, including Zig-Zag chains and Y-shape assemblies, by simply altering the spatial geometries of microchannel templates. In general we have developed a robust microfluidic platform for the construction of complex microassemblies with multiple geometries functionalities, which can serve as futuristic methods of “bottom-up” assembly and should be of interest in various fields like microfabrication, microfluidics, and biomimics.
10:15 AM - II4.3
BioMEMS for Growth of Endothelial Cells.
Susmi Das 1 2 3 , Fatima Merchant 3 , Wanda Zagozdzon-Wosik 1 Show Abstract
1 Electrical and Computer Eng., University of Houston, Houston, Texas, United States, 2 Texas Center for Superconductivity, University of Houston, Houston, Texas, United States, 3 College of Technology, University of Houston, Houston, Texas, United States
The in-vitro growth of endothelial cells (ECs) to form blood vessels is increasingly important in tissue engineering, but the mechanisms governing vasculogenesis are still poorly understood. Results from previously reported studies which have evaluated the influence of substrate material properties such as mechanical, chemical and electrical, topology and topography, and various environmental cues using 2D, 3D as well as sheet based configurations, are difficult to reconcile due to inconsistencies originating from the varied but specific experimental conditions implemented. We have designed a BIOMEMS device for controlled EC growth, which provides mechanical support and specific patterns that facilitate vascular network formation. We utilized various materials fabricated both as 2D and 3D structures in matrices of different geometries, to determine factors that affect cell growth and proliferation. For fabrication, we adapted Si technology and implemented layers of silicon oxide, nitride, and borides. Layers were either grown (thermal silicon oxide) or deposited using Chemical Vapor Deposition (nitride) and e-beam evaporation (borides). Detailed material characterization was done for all layers. Pattering was by optical lithography (Futurrex photoresists) followed by etching using wet and/or dry processes. Lines were patterned in sizes ranging from 5 to 60 µm shapes to mimic vascular networks including capillaries. The geometry and proximity of the patterns as well as their material dependent layer properties and surface passivation, facilitated inter-cellular interaction during the process of adhesion, spreading and locomotion. Next, to observe the contact guiding effect on cell growth in 3D structures, we fabricated grooves in Si using the same 2D patterns where these effects were identified. We used etching in KOH to form V- and trapezoidal grooves 5μm to 50μm wide and 3.5μm to 25μm deep. In selected experiments, grooves were gelatin coated to direct and promote cell alignment and proliferation. Human umbilical vein endothelial cells (HUVEC) were cultured to confluence, split and seeded on the cleaned and sterilized substrates for culturing. Cell cultures were maintained at 37°C with 5% carbon dioxide. The cultured cells were observed via transmitted light, and fluorescence confocal microscopy using viability dyes (5mM acridine orange). HUVEC showed selective contact guidance dependent on material properties, pattern geometry, and surface preparation. Adhesion, elongation, and growth of ECs and their proliferation were obtained on Si3N4 and borides but not on SiO2 or Si. Cellular interaction and formation of a monolayer of vascular networks on the substrate was observed. On the Si substrate with grooves, the cells aligned and were mechanically interlocked in the grooves; and exhibited elongation and growth.
10:30 AM - **II4.4
Chemical Engineering-Based Multiscale Optimization of 3D Cellular Organization and Oxygen Supply In Vitro.
Yasuyuki Sakai 1 Show Abstract
1 Institute of Industrial Science, University of Tokyo, Tokyo, Tokyo, Japan
Our main concern is the 3D organization of cultured organ-derived cells such as liver cells in various scales for regenerative medicine and cell-based assay for drug or chemical screenings. In our body, 1) Cells are hierarchically organized at a very high cell density, but 2) The vascular system consistently supplies nutrients and removes waste/metabolites, thus attaining very high per-volume-based functionality. However, arrangement of such functional vascular systems in vitro is still a very difficult issue and it thus becomes a serious problem to simultaneously optimize 3D high-density cellular organization and to secure good mass transfer between the cells and culture medium. Chemical engineering-based analyses, design of tissues, and integration of suitable technologies are very helpful in addressing the problem in various scales. When we really intend to organize large tissue equivalents for implantation therapy, the tissue should at least be arranged with a 3D branching/joining flow channel network as an in vivo vasculature and the channels should be perfused with suitable culture medium containing oxygen carriers. We proposed a design criteria based on oxygen diffusion-consumption around a flow channel in macroporous 3D scaffolds, fabricated them, and evaluated their efficacy and limitation in perfusion culture of liver cells. Also, we checked the feasibility and problems of existing hemoglobin-based oxygen carriers. When we intend to make a small tissue for cell-based assays, we probably do not need to arrange vasculature, but we have to organize the cells to a certain extent and create an in vivo-mimicking micro-environment. We again need to pay a special attention to mass transfers between the organized cells and the culture medium. As one of the solutions, we proposed direct oxygenation through highly oxygen-permeable polydimethylsiloxane (PDMS) membranes to solve completely the limitation of oxygen supply to liver-derived cells in static culture. In particular, we are stressing that meeting the cellular oxygen demand at appropriate physiological concentrations enables highly-efficient aerobic respiration of the cells with less oxidative stresses, leading to spontaneous 3D cellular organizations that have never been observed before in vitro. As such, focusing on oxygen supply to the cells should give a firm basis for the design of culture systems in various scales for various applications.
11:30 AM - **II4.5
Fabrication of Complex Hydrogel Materials by Utilizing Microfluidics and Micromolding.
Masumi Yamada 1 , Yoji Naganuma 1 , Emi Yamada 1 , Shunta Kakegawa 1 , Sari Sugaya 1 , Minoru Seki 1 Show Abstract
1 Applied Chem. & Biotechnol., Grad. Sch. of Eng., Chiba University, Chiba, -, Japan
Microfluidic and microfabrication techniques are being developed for producing functional biomaterials for tissue engineering applications. We have proposed microfluidic systems to produce hydrogel scaffolds for cell culture, having various shapes including particles, fibers, sheets, thin patterns, and microfabricated plates. For example, we have demonstrated the continuous and rapid production of calcium alginate gel fibers with diameters of 1-200 micrometer, by using a microfluidic device, and the produced fibers were strong enough to roll around longer than 100 m. Several researchers have reported on the synthesis of Ca-alginate or chitosan hydrogel fibers either by using a micro-nozzle or a double capillary. However, it was impossible to obtain complex hydrogel fibers composed of different components in the cross section when conventional methods are employed. We have proposed a microfluidic system for synthesizing Ca-alginate hydrogel fibers composed of hard and soft regions, which enables the guided cell growth along the fiber direction. By introducing sodium alginate solution, buffer solution, and the gelling solution of CaCl2 into a co-flowing microchannel, and by controlling the flow rates and the microchannel geometries, we successfully fabricated anisotropic microfibers with diameter from 5 to 200 micrometer. Also, we employed propylene glycol alginate (PGA) to make a soft core sandwiched by solid shells, and tried to guide the direction of cell growth. As a result, 3T3 or HeLa cells, initially located in the soft core, grew along the soft region and formed linear colonies after several days of cultivation. In addition, we successfully controlled the direction of neurite elongation, and formed cell-cell networks between multiple PC12 cells. The hydrogel fibers with the anisotropic cross-sectional morphologies are promising as a material for enabling guided growth of various kinds of cells and can be used as functional parts for 2D/3D cell assembly for tissue regeneration or transplantation.
12:00 PM - **II4.6
3D Cell Co-Culture System on Hydrogel Micro-Patterned Surface.
Keitaro Yoshimoto 1 Show Abstract
1 , The University of Tokyo, Tokyo Japan
The recent progress in the combination of cell culture and microfabrication technologies has stimulated the research on the development of new methods of cell culturing on chips for medical purposes. Especially, the high-performance cell culture to control cell’s functions, such as retention of viability, activity, differentiation and proliferation, is more required in the field of regenerative medicine. In order to realize controllable cell culture for regenerative medicine, it is also important that the in vivo-like culture is fabricated. So we focus on a spheroid co-culture system for hepatic cells and primary hepatocytes. The method for constructing multicellular spheroid plays many important roles in various metabolic pathways and express in vivo-like function, respectively. The micropatterned PEG-gel chip was prepared on glass surface by photolithography technique. In this system, in order to construct feeder-cell micropatterned surface before fabrication of spheroid formation, bovine aorta endothelial cells (BAECs) and non-parenchymal cells (NPCs) were seeded on the constructed PEG-gel patterned surface as single layer. Among the hepatic cells, fetal mouse liver cells (FMLCs) have been studied as a new material for growing artificial livers, liver-cell implantation, and served as a model of undifferentiated hepatocytes in the adult liver. FMLCs are regarded as a suitable cell source for implantation and regeneration due to their genetic normality and potentially proliferative activity in vitro. In this study, we tried to fabricate FMLCs spheroids arrays on micropatterned PEG-gel surface chip and evaluate the activity of the FMLCs and the efficiency of the differentiation induction. FMLCs spheroid were cultivated by seeding FMLCs with various cell concentrations onto the constructed BAECs and NPCs micropatterned surfaces. 10 ng/mL of oncostatin M (OSM), which is differentiation induction for matured liver cell, was added to the incompletely formed spheroid array on day 1 after seeding FMLCs. To assess the liver function of the cultured FMLCs spheroids, albumin secretion was quantified by a sandwich enzyme linked immune sorbent assay as activity of hepatocyte, and CYP450 1A2 activity as differentiation marker of matured hepatocyte was measured using chemiluminescence intensity from luciferin. As results, we succeeded in constructing a two-dimensional array of FMLC spheroids on a micropatterned PEG-gel surface chip. Interestingly, the spheroids did not only show the long viability and high albumin secretion, but also high degree of differentiation induction. This novel co-culture system based on cell-chip technology could provide an interesting new approach for cell culture system for primary, cancer, and stem cells.
12:30 PM - II4.7
Electrohydrodynamic Jet Printing for Hydrogel Cell Culture Substrates.
Michael Poellmann 1 , Kira Barton 2 , Amy Wagoner Johnson 3 Show Abstract
1 Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 2 Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 3 Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States
Chemical and physical factors in the cellular microenvironment strongly influence, if not direct, many aspects of cell behavior. The development of in vitro microenvironments with precise control over adhesion ligands, geometry, and mechanical properties is critical to understanding this regulation. In this work, we introduce a method to pattern polyacrylamide substrates with Electrohydrodynamic Jet (E-Jet) printing. E-Jet printing is a method for patterning features at the micro- and nanoscale. Ink is pushed through a glass capillary tube with applied back pressure, then pulled into a conical meniscus by an electric field. Droplets that jet from the tip of this Taylor cone-shaped meniscus result in spot sizes significantly smaller than inkjet printing. This work represents the first application of E-Jet printing on a soft substrate, in this case, polyacrylamide-co-acrylic acid functionalized with N-hydroxysuccinimide. These hydrogels are architecturally and mechanically similar to soft tissue, and are designed to efficiently form covalent bonds to printed proteins. Non-printed regions are deactivated in subsequent rinsing steps. Substrates are patterned with an easily-detectible protein, IgG, and an extracellular matrix protein, fibronectin. Patterns are printed using drop-on-demand mode to create arrays of equally-spaced spots, continuous jet mode for straight lines, and pulsed mode for high-speed printing. We demonstrate patterns with spot diameters smaller than 5 µm, which compares favorably to microcontact printing. Compared to stamping methods, E-Jet offers much higher flexibility, with the ability to change patterns at the point of printing. Fibronectin-patterned substrates with Young’s moduli ranging from 2 to 65 kPa are shown to support the adhesion, proliferation, and differentiation of mesenchymal stem cells (MCSs), a cell type separately known to be sensitive to substrate stiffness and to adhesive geometry. Such substrates are being used to study how adhesion geometry, chemistry, and substrate stiffness interact to influence the differentiation of MSCs into osteoblasts.
12:45 PM - II4.8
Toward a Lithographically Patterned Bio-Artificial Pancreas.
Jaehyun Park 1 , Yevgeniy Kalinin 1 , Christina Randall 1 , David Gracias 1 Show Abstract
1 , Johns Hopkins University, Baltimore, Maryland, United States
We describe the use of lithographic processes to precisely structure a three dimensional bio-artificial pancreas from the nano to the macro scale. This precision is combined with the utilization of novel self-assembly and molecular surface modification methods to create a device that facilitates adequate diffusion to transplanted islet cells while also enabling immunoisolation. Using both simulations and experiments, we investigate (a) architectural constraints that minimize dead or hypoxic zones; (b) the influence of nanopores in enabling size-exclusion based immunoisolation; and (c) insulin release from devices with encapsulated islet cells.
II5: BioMEMS Tools for Cell Mechanics
Tuesday PM, November 29, 2011
Room 206 (Hynes)
2:30 PM - **II5.1
Implementation of BioMEMS for Determining Mechanical Properties of Biological Cells.
Svetlana Tatic-Lucic 1 , Markus Gnerlich 1 Show Abstract
1 ECE, Lehigh University, Bethlehem, Pennsylvania, United States
Even though microelectromechanical systems (MEMS) have been researched and developed for several decades now, only relatively recently has their full potential in the fields of medicine and biology been recognized and exploited. Within that framework, their applications are particularly attractive in cell biology, because of the nearly perfect compatibility of their sizes, as well as the simultaneous flourishing of both miniaturization science and bioengineering.Determining mechanical properties of biological cells, which was a challenge to do because of the lack of techniques that would be capable of executing this task accurately and efficiently, and on more than one individual cell at the time, proved to be a very fruitful BioMEMS targeted application. In this paper we are reporting on our recent work to determine the mechanical properties of biological cells using a BioMEM system based on an electrostatic actuator with predetermined step-wise deflection, piezoresistive force sensor, temperature sensor for measuring and heater for regulating the temperature of the cell medium, as well as a dielectrophoretic trap for positioning of the cells. There is a number of challenges associated with this system: 1) all of its elements need to be functional in a conductive liquid such as cell medium, 2) the force sensor needs to be extremely sensitive (forces that need to be measured are below 100nN), 3) temperature of the cell medium has to be maintained close to 37degC, and not be elevated during the actuating sequence. We will discuss the material issues, design and characterization details of this system. We will also discuss the results of initial cell mechanics experiments.
3:00 PM - II5.2
The Use of Controlled Surface Topography and Flow-Induced Shear Stress to Influence Renal Epithelial Cell Function.
Else Frohlich 1 2 , Xin Zhang 2 , Joseph Charest 1 Show Abstract
1 Bioengineering, Draper Laboratory, Cambridge, Massachusetts, United States, 2 Mechanical Engineering, Boston University, Boston, Massachusetts, United States
Physiologically-representative and well-controlled in vitro models of human tissue provide a means to safely, accurately, and rapidly develop therapies for disease. Applying mechanical cues, such as sub-micron substrate topography and flow-induced shear stress (FSS), can control cell functions such as alignment, migration, differentiation and phenotypic expression of cells [1, 2]. Leveraging these effects, we combined and independently controlled topography and FSS in a cell culture device to control cell function resulting in a physiologically-representative in vitro model of human tissue. The microscale tissue modeling device (MTMD) coupled an embossed topographical substrate with a molded microfluidic chamber to control both topography and FSS independently. The topographical substrate possessed surface features consisting of 750 nm wide ridges and grooves, generated using a hot-embossing mold that was fabricated via a unique optical lithography, etch, and electroforming process. As a renal cell model, cells from the human renal proximal tubule cell line HK-2 were cultured in the MTMD and exposed to user-defined topography and various FSS levels for two hours. Tests were conducted using both blank and topographical substrates, allowing the effects of FSS and surface topography to be studied independently and simultaneously. Results show that topography and FSS work in concert to elicit cell alignment and influence tight junction (TJ) formation. Cells aligned when presented with both topography and FSS, with alignment levels increasing further as FSS levels were increased. Formation of robust TJs, as measured by ZO-1 intensity and continuity around cell perimeters, increased for cells on topographic substrates. FSS further enhanced the robust TJ formation of cells on topographic substrates. As these alignment and TJ formation changes occurred more rapidly than in previous studies, the topographic patterns may have enhanced or accelerated renal proximal tubule response to FSS, demonstrating the value of the combination of these two mechanical cues. The MTMD provides a more realistic in vitro model of human kideny tissue by administering independently-controlled mechanical cues of topography and FSS to cell populations. The platform shows great promise to enhance cell function studies, speed drug development, and provide a pathway to regenerative medicine therapies.References:  Teixeira AI. Biomaterials. 2006:3945-3954.  Dalby MJ. Nature Mat. 2007: 997-1002.
3:15 PM - II5.3
Fabrication and Characterization of a Polymeric Microdevice for Cell Loading with Controllable Strain Distribution.
Qian Wang 1 , Yi Zhao 1 Show Abstract
1 Biomedical Engineering Department, Ohio State University, Columbus, Ohio, United States
Live cells are constantly subjected to mechanical signals, which are critical for regulating cellular functions under various physiological conditions. To quantitatively understand the effects of these mechanical signals, many engineered methods are developed for applying mechanical loads to cells. Among these methods, applying mechanical strains by deforming thin polymeric membranes is widely used. Nonetheless, this method is primarily used at conventional scale where a large number of cells are strained at the same time. Straining a selected group of cells in culture, which is essential for intercellular mechanotransduction study, however, is not yet implemented.This paper reports development of a microdevice that can deliver controllable bi-axial mechanical strains to selected cells in culture. To address the non-uniform strain field in microscale polymer membranes, a microfabrication strategy is developed to tune the strain gradient. With the ease of mechanical stimulation and the controllable strain gradient, this work promises a potential in quantitative intercellular mechanotransduction study.Similar as the counterparts at the conventional scale, mechanical strains are applied to cells by deforming a polymeric membrane. Thin polydimethylsiloxane (PDMS) membranes are fabricated using soft-lithography and connected with microfluidic channels. Each PDMS membrane is 500μm in diameter and 60μm in thickness. Upon actuation, fluid in the microchannels deforms the PDMS membranes and delivers controllable strain to cells. Each membrane is independently deformable so that strain with desired magnitudes can be delivered independently to each membrane. However, since the PDMS membrane has a constant thickness, a highly non-uniform strain profile is generated upon a differential pressure. Cells on the membrane are thus subjected to different magnitudes of strain. This may complicate the subsequent analysis. Finite element analysis shows that strain gradient can be controlled by designing PDMS membrane with varying thickness. For example, a membrane with a large thickness in the center and a small thickness in the peripheral area can lead to a more uniform strain profile. Such a membrane with a varying thickness is fabricated by using a deformed PDMS membrane as the master template mold and transferring the deformed profile to a second PDMS substrate using soft-lithography. After fabrication, the actual strain profile upon actuation is experimentally determined by monitoring the displacements of the microdots patterned on the membranes. The results agree well with the finite element analysis, where the membrane with a constant thickness has a large strain gradient, and the membrane with a varying thickness exhibits a small strain gradient change in the majority area of the membrane. This work provides a starting point for designing more complicated strain gradient profile for cellular mechanotransduction studies.
3:30 PM - **II5.4
Opto-Mechanical Platforms for Cell Force Study.
Xin Zhang 1 Show Abstract
1 Mechanical Engineering, Boston University, Boston, Massachusetts, United States
Microsystems are providing key advances in studying single-cell mechanical behaviors. The mechanical interaction of cells with their extracellular matrix is fundamentally important for cell migration, division, phagocytes and apoptosis. As the displacement and scales of cellular phenomena is comparable to optical wavelength, optical metrology offers superior resolution and real-time imaging capabilities to measure cell forces and subcellular behavior as compared to its traditional counterparts. In this talk, I will present new advances in cellular force measurement based on opto-mechanical-based methods and discuss its unique capacities in studying cell mechano transductions.
4:30 PM - **II5.5
Microtechnologies for Studying Cell Mechanobiology.
Craig Simmons 1 2 Show Abstract
1 Department of Mechanical & Industrial Engineering, University of Toronto, Toronto, Ontario, Canada, 2 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
Cells reside in three-dimensional, soft extracellular matrices where they interact with other cells and, in the case of cardiovascular and musculoskeletal tissues, are subjected to dynamic mechanical loading. However, in traditional cell culture platforms (e.g., microtiter well plates), cells are grown on rigid, static two-dimensional surfaces. Thus, current platforms for studying cardiovascular and musculoskeletal cell biology poorly represent the in vivo environment, which limits the novelty and translatability of the biological information they generate. In this talk, I will describe some of the microtechnologies that we are developing to address these limitations. These microfluidic platforms are designed to allow precise control over the cellular microenvironment, including matrix stiffness and proteins, soluble proteins, cell-cell interactions, and biophysical forces. Compared with standard culture platforms, these microsystems better mimic key components of the in vivo cellular mechanobiological microenvironment, offer more precise control over microenvironmental cues, and avoid some of the confounding factors associated with application of mechanical forces at the macroscale. They are also compatible with on-chip imaging and are highly parallelizable, and therefore can be used in high-content and high-throughput screening applications. Current applications include fundamental studies of cell-matrix and cell-cell interactions in mechanically active environments and screening of biomaterial properties for stem cell-based tissue regeneration.
5:00 PM - **II5.6
Wetting Phenomena of Phospholipid Films for Electroporating Membranes.
Evelyn Wang 1 , H. Jeremy Cho 1 , Shalabh Maroo 1 Show Abstract
1 Mechanical Engineering, MIT, Cambridge, Massachusetts, United States
We investigated wetting phenomena of phospholipid films for the development of tunable electroporating membranes. Electroporation is widely used to permeabilize the cell membrane with an electric field for applications such as DNA transfection, gene therapy, and targeted drug delivery. More recently, electroporation has received interest to actively control artificial membranes made of supported lipid bilayer films for cell and chemical separations in bioMEMS applications. To successfully achieve such control, however, a thorough molecular understanding of lipid-fluid interactions is needed. In this work, we experimentally quantified surface energies associated with the tail of DPPC lipid monolayers on mica and glass substrates. We studied the quality of these films, orientation of lipids, and wetting behavior at varying surface pressures. The surface morphology of these films was characterized using atomic force microscopy. Three different probe liquids of varying polarity, water, formamide, and diiodomethane, were used. Advancing contact angles ranging from 70o to 100° were obtained for all fluids. However, the high contact angle hysteresis (~30-50°) and large effect on adhesional stability of the films suggests the strong interaction between polar liquids (water and formamide) and the hydrophilic substrates (mica and glass). Meanwhile, the minimal hysteresis (~10°) and little change in surface morphology indicates the weak interaction between the nonpolar diiodomethane and mica. To facilitate understanding of these experimental results, molecular dynamics simulations were performed on supported DPPC monolayers, which suggest how changes in film morphology occur at different surface pressures. This work offers a first step towards quantifying surface energies of different lipids and providing the molecular understanding necessary to tune pore size in electroporating membranes.
5:30 PM - II5.7
On-Chip Measurements of Cell Compressibility Using Acoustic Standing Wave.
Deny Hartono 1 , Yang Liu 2 , Kian-Meng Lim 2 3 , Lin-Yue Lanry Yung 1 Show Abstract
1 Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Singapore, 2 Singapore-MIT Alliance, National University of Singapore, Singapore Singapore, 3 Department of Mechanical Engineering, National University of Singapore, Singapore Singapore
Measurements of mechanical properties of biological cells are of great importance for investigating different stages of cell differentiation, for examining health status of cells and for facilitating cell separation. Up to date, however, direct measurements of bulk modulus or compressibility of cells are very limited. Herein, we report on-chip measurements of the compressibility of a series of cell lines and water-borne parasites using acoustic radiation for gaining further insights on the mechanobiological meaning of cell compressibility. To achieve this, we have developed a new class of acoustic-based tool that permits fast and continuous cell compressibility measurements. The tool exploits the formation of acoustic standing wave within a straight microchannel. When polystyrene beads and cells are introduced into the channel, the acoustic radiation force moves them to the acoustic pressure node. By fitting the experimental and theoretical trajectories of the beads and the cells, the acoustic energy density and subsequently the compressibility of the cells can be obtained respectively. We find that the co