Symposium OrganizersMichael Schwartz, University of Wisconsin-Madison
Todd C. McDevitt, Georgia Institute of Technology
Matthias P. Lutolf, EPFL-SV-IBI-LSCB
Joel P. Schneider, National Cancer Institute-Frederick
Symposium Support Glycosan Biosystems, Inc. A Division of Orthocyte Corp.
National Science Foundation
PP2: Engineering Platforms for Dynamic or Spatial Control Over the Microenvironment
Tuesday PM, April 10, 2012
Marriott, Yerba Buena, Salons 10-11
2:30 AM - *PP2.1
3-D Biofabrication for Development of Cellular Systems
Piyush Bajaj 1 2 Vincent Chan 1 2 Jae H Jeong 4 5 Pinar Zorlutuna 1 2 Chaenyung Cha 4 Hyun J Kong 4 2 5 Rashid Bashir 3 1 2
1UIUC Urbana USA2UIUC Urbana USA3UIUC Urbana USA4UIUC Urbana USA5UIUC Urbana USAShow Abstract
Engineering complex 3-dimensional tissues presents great promises to improve treatment of tissue defects and to provide better understanding of emergent behavior of normal and pathologic cells. Hence, there is an immediate need for systems that can recapitulate and further manipulate the tissues of interest, in a high throughput and automated manner while maximizing the cell and tissue functionality. Such approaches can also be used to develop cellular systems and to study the emergent behavior of integrated cellular systems. In this talk, we will present an overview of our work using 3-D stereolithography to develop polymeric structures embedded with live cells for a range of applications. Firstly, we show that cell-encapsulated hydrogels with complex three-dimensional (3D) structures can be fabricated from photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) using modified â?~top-downâ?T and â?~bottoms-upâ?T versions of a commercially available stereolithography apparatus (SLA). Long-term viability of encapsulated NIH/3T3 cells was quantitatively evaluated using an MTS assay and shown to improve over 14 days by increasing the Mw of the hydrogels. Secondly, we show that we can spatially organize primary hippocampus neurons (HNs) and skeletal muscle myoblast cells (MCs) in a 3D hydrogel matrix with tunable mechanical and degradation properties. The spatial organization of these multiple cell types revealed that the presence of MCs resulted in increased cholinergic functionality of the HNs, as quantified by their choline acetyltransferase activity. And finally, we show that a â?~livingâ?T microvascular stamp can be fabricated which releases multiple angiogenic factors and subsequently creates neovessels with the same pattern as that engraved in the stamp. The microvascular stamp developed in this study would serve to direct the emergent cellular behavior towards vascularization, improve the quality of revascularization therapies, and allow the vascularization of biological machines in-vitro.
3:00 AM - PP2.2
Microfluidic Cell Culture Chambers with Nanoporous Walls for Chemical Communication
Zhifei Ge 1 Peter R Girguis 2 Cullen R Buie 1
1Massachusetts Institute of Technology Cambridge USA2Harvard University Cambridge USAShow Abstract
Reconstruction of phylogenetic trees based on 16S rRNA gene sequencing reveals that so far only a tiny fraction of microbial diversity has been cultured in the laboratory. One major reason behind this â?ounculturabilityâ? is that many microbes function in symbiosis, frequently exchanging metabolites to sustain their own metabolism, while key exchanged metabolites have hardly been identified. To advance the culturability of diverse microbes we propose a method to engineer a microfluidic co-culture platform that mimics natural conditions for bacterial growth. The key to innovation is to physically isolate bacteria while allowing chemical communication through metabolite diffusion. In our method, we use a porous material, poly(2-hydroxyethyl methacrylateco-ethylene dimethacrylate) (HEMA-EDMA), to fabricate a microwell array with 105 individual culture chambers. Pore size of HEMA-EDMA was confirmed by ESEM imaging to be less than 200 nm, adequate for isolating all identified bacteria. We have video-recorded fluorescence labeled Escherichia coli swimming in confined of HEMA-EDMA wells and observe that E. coli is unable to go travel between culture chambers. In our culturing process, we stochastically seed fluorescently-labeled E. coli into each 1 nanoliter sized well with prescribed cell solution, a 500 times dilution of the E. coli culture solution in stationary phase. Our statistical results of 300 wells showed that on average 0.92 cells were seeded in each well with a standard deviation of 0.79. In order to prevent evaporation of growth media, we packaged the device with a PDMS cap and immersed the device in water. After about 36 hours of cell culture at 30Â°C without refreshment of growth media, 53.7% of the seeded wells were observed to have exponential bacterial growth. In this case only one well with observed growth was not initially seeded. Suggesting that cross-contamination by way of â?oswimmingâ? from one well to neighboring wells was minimal. In this presentation we will explore co-culture of bacterial species which rely upon a syntrophic relationship in our novel device. This will be demonstrated via symbiotic strains of E. coli and Salmonella genetically engineered to exchange lactate to ensure survival. In the future we will utilize this device to search for previously uncultivable microbes that rely upon syntrophic relationships.
3:15 AM - PP2.3
Deconstructing the Effects of Matrix Stiffness and Confinement on Cell Migration
Amit Pathak 1 Sanjay Kumar 1
1University of California, Berkeley Berkeley USAShow Abstract
Cell migration is a dynamic process strongly regulated by biophysical interactions between cells and the extracellular matrix (ECM). These mechanosensitive interactions are driven by sub-cellular mechanisms including protrusions at the leading edge of the migrating cell, adhesions with the ECM, and actomyosin contractility, all of which depend on both the stiffness and geometry of the extracellular matrix (ECM). While the influence of ECM stiffness on cell migration, adhesion, and contractility have been extensively studied in two-dimensional ECMs, extension of this concept to three-dimensional ECMs that more closely resemble tissue has proven challenging, because perturbations that change matrix stiffness often concurrently change matrix porosity. This convolution of ECM biophysical properties is particularly problematic given that pore size has been independently demonstrated to regulate migration speed. Here we investigate this problem using a novel microscale culture platform and mathematical modeling. First, we introduce a microchannel-based matrix platform that allows orthogonal variation of ECM stiffness and channel width. For a given ECM stiffness, cells confined to narrow channels surprisingly migrate faster than cells in wide channels or on unconstrained 2D surfaces, which we attribute to increased polarization of cell-ECM traction forces. Confinement also causes migration speed to increase monotonically with ECM stiffness, in contrast with the biphasic relationship observed on unconfined ECMs. We attribute this effect to the fact that channel confinement forces polarization of traction force, which in turn enhances fast, persistent migration. Inhibition of nonmuscle myosin II dissipates this traction polarization and renders the relationship between migration speed and ECM stiffness comparatively insensitive to matrix confinement. To integrate all of these hypotheses into a coherent, quantitative framework, we develop a predictive multiscale model of a cell migrating in an ECM channel of defined width and stiffness, which we show recapitulates key experimental trends. These studies introduce a new paradigm for investigating matrix regulation of invasion and demonstrate that matrix confinement modulates the relationship between cell migration speed and ECM stiffness.
3:30 AM - PP2.4
Flipping the Switch: Engineering Cell-instructive Microenvironments with Photo-activatable ``Caged'' Peptides
Daniel L Alge 2 Kristi S Anseth 1 2
1University of Colorado Boulder USA2Howard Hughes Medical Institute Boulder USAShow Abstract
Photochemical patterning is a powerful tool for manipulating the biochemical composition of extracellular matrix mimetic hydrogels. The general approach has been to swell in a biomolecule such as a peptide, and then conjugate it to the hydrogel matrix via a photo-initiated reaction. Excellent 2D patterning fidelity can be achieved with this method by using conventional photomasking technology, and complex 3D patterns can be created by two-photon irradiation. It is even possible to create complex microenvironments with multiple biomolecules by sequential patterning. Nevertheless, the ability of the soluble biomolecule to interact with cells during the swelling process is a potential limitation. To address this problem, we propose here the use of a biomolecule caging strategy. In short, biomolecule caging is the general approach of modifying a biomolecule with a photo-labile moiety to temporarily render it biologically inactive, but photo-activatible. Thus, caging is also a photochemical approach, and the same techniques for hydrogel patterning described above can be utilized. However, because the molecule must be irradiated and â?oswitched onâ? to become bioactive, this approach circumvents the potential for unwanted interactions with cells outside of the patterned region of the hydrogel. Importantly, caging has shown exceptional utility in biochemistry studies because it allows for user-controlled signal activation with spatio-temporal resolution. Numerous caged biomolecules have been described in the literature including morpholinos, oligonucleotides, peptides, and even proteins. Nevertheless, biomolecule caging has only been used minimally in biomaterials and tissue engineering. To show proof of concept, we have designed caged RGD peptides that can either be incorporated into the hydrogel matrix during initial gel formation, or patterned in at a later time point with an orthogonal photo-initiated reaction. RGD caging with nitrobenzyl and amino coumarinyl moieties, which have the potential for orthogonal photo-cleavage, was explored. Solution studies of uncaging showed that the proper irradiation conditions resulted in efficient uncaging to produce the bioactive RGD peptide without side reactions. Our efforts to dynamically regulate cell-matrix interactions using these caged RGD peptides will be presented.
4:15 AM - *PP2.5
Dynamic Cell Niches through Bioorthogonal Photochemical Reactions
Cole DeForest 1 Mark Tibbitt 1 Kristi Anseth 1 2
1University of Colorado Boulder USA2Howard Hughes Medical Institute Boulder USAShow Abstract
There is a growing interest in the development of novel biomaterial platforms that can be tuned both dynamically and in real time to probe the effects of the surrounding materials on cell function. Taking lead from the recently popularized â?oclickâ? philosophy, where chemical reactions proceed efficiently and with high specificity, orthogonal reactions can be exploited to control different physical and chemical aspects of cell-laden polymer scaffolds. This contribution will detail one such synthetic platform to engineer multifunctional networks based on three orthogonal reactions that enable user-defined biochemical and biomechanical cues to be introduced in the presence of cells at any point in time and space. Gels are first formed via a copper-free click reaction between azide and cyclooctyne moieties, enabling the encapsulation of cells in idealized, initially-uniform networks. Subsequently, a thiol-ene photocoupling reaction is introduced that enables patterning of biological functionalities within the gel, allowing one to tailor the chemical properties of the cell culture niche in situ. Finally, a photodegradable nitrobenzyl ether is incorporated into the hydrogel backbone that allows real-time manipulation of mechanical properties of the system. As both the photocoupling and photodegradation reactions are initiated with different wavelengths of light (365 and >500 nm), they can be performed orthogonally, as verified with NMR, and with full spatial and temporal control illustrated in 3D. This presentation will detail our efforts towards network characterization of chemical functionalization and degradation with 3D confocal microscopy, profilometery, and rheometry. Further, results will be presented to highlight the versatile nature of the chemistry to create programmable niches to study and direct human mesenchymcal stem cell function and mouse embryonic stem cell pluripotency by modifying the local hydrogel environment.
4:45 AM - PP2.6
Engineering Stem Cell Microenvironment with Independently Tunable Biochemical and Mechanical Properties
Xinming Tong 1 Fan Yang 1 2
1Stanford University Stanford USA2Stanford University Stanford USAShow Abstract
Introduction: Scaffolds with properties that mimic the natural extracellular matrix is highly desirable for regulating stem cell fate and promoting tissue regeneration. Conventional 3D biomaterials are unable to vary biochemical and mechanical cues independently. For example, increasing collagen increases cell adhesion density but also changes the mechanical stiffness of the resulting network. To address these limitations, the goal of this study is to develop an interpenetrating network which allows independent tuning of the biochemical and mechanical properties of the hydrogel. To construct two polymer networks simultaneously and independently, two different crosslinking mechanisms were utilized, namely radical polymerization of methacrylate and coupling of amine with N-Hydroxysuccinimide ester (NHS). Both approaches can be carried out at physiological conditions and are cell-friendly. Materials and Methods: We chose poly(ethylene-glycol) (PEG) as the backbone structure due to its â?oblank slateâ? structure and amenability to chemical modification. To introduce pendent modifiable sites, we propose to perform condensation of PEG, then introduce methacrylate end group to make it photocrosslinkable, followed by pendent NHS introduction and peptides incorporation. Peptides derived from both insoluble ECM or soluble growth factors were conjugated. The mechanical network was constructed from linear amine terminated PEG (molecular weight variable) coupling with 4-arm NHS terminated PEG (Mw~10k). The linear PEG component was synthesized by coupling carboxylic terminated PEG with MMP-1 sensitive peptide sequence GPQGâ?"IWGQK to make the network enzymatically degradable, or by coupling carboxylic terminated PEG with PEG diol to introduce ester linkage into the backbone and make it hydrolytically degradable. The degradation rate can be tailored by changing the concentration and type of the degradable linkages. Results and Discussion: Using a modular design approach, we have developed an interpenetrating network with two distinct crosslinking mechanisms for the biochemical and mechanical â?obuilding blocksâ?. We have successfully synthesized the precursor polymers with final yield higher than 70% and the structures of newly synthesized polymers were verified by NMR. The biochemical precursor with pendent carboxylic groups formed hydrogel upon light exposure (365nm, 5mW cm-2) using I2959 as initiator. The mechanical precursor polymer with amine terminated groups formed hydrogel with 4-arm NHS terminated PEG, and the mechanical strength can be varied across a broad range by changing the number-average molecular weight between crosslinks (Mc) and the concentration of the polymer. The technology platform is very versatile and can be adapted to optimize niche cues for directed differentiation of any type of stem cells towards any lineages.
5:00 AM - PP2.7
Manipulating Thiol-ene Hydrogel Degradation for Controlling 3D Cell Morphogenesis
Han Shih 1 Chien-Chi Lin 1
1Indiana-University Purdue-University Indianapolis Indianapolis USAShow Abstract
An ongoing effort in tissue engineering is to design hydrogels with tunable degradability for controlled release and cell encapsulation studies. Recently, hydrogels prepared from step-growth thiol-ene photochemistry have been used to encapsulate a variety of cell types for tissue regeneration, including pancreatic beta cells and human mesenchymal stem cells (hMSCs). In this gelation scheme, norbornene acid functionalized 4-arm poly(ethylene glycol) (PEG) was crosslinked, via a thiol-ene photo-click reaction, with bis-cysteine terminated peptides to form PEG-peptide hydrogels. Due to the presence of an ester bond between the cyclic olefin and PEG, these hydrogels degrade hydrolytically via ester hydrolysis. Furthermore, these thiol-ene hydrogels may be degraded enzymatically with the use of enzyme cleavable peptides as hydrogel crosslinkers. We have proven that the degradation characteristic can be â?~switchedâ?T from surface erosion to bulk degradation through modulating peptide crosslinkers with different enzyme sensitivities. We also synthesized a series of thiol-ene hydrogels with different peptide crosslinker sequences and functionalities, from which to tune the time required for complete gel degradation (from weeks to months in vitro). In addition, we utilized a statistical-co-kinetic model to predict the degradation profiles of these thiol-ene hydrogels. Specifically, this model takes into account the variations in hydrogel crosslinking efficiency, hydrolysis kinetics, as well as polymer structural information (including molecular weight and functionality of the macromer and crosslinker). While the degradation of ester-linked thiol-ene hydrogels is base-catalyzed, our experimental results indicated a slower than predicted degradation rate at basic condition (pH8), indicating the existence of another base-catalyzed mechanism within the hydrogel network that retards gel degradation. We hypothesized that this phenomenon was due to the oxidation of thioether bonds between PEG macromer and thiol-terminated crosslinker. The benefits of highly tunable and predictable thiol-ene hydrogel degradation will be realized via controlled cell morphogenesis in 3D.
5:15 AM - PP2.8
Influence of a Zwitterionic Polymer Brush on Stem Cell Morphology and Function
Aftin Monique Ross 1 Thomas Eyster 2 Luis G Villa-Diaz 3 Jonathan Oh 4 Priya Moni 4 Domenic Kratzer 5 Paul H Kresbach 1 3 Joerg Lahann 1 2 4
1Univ Michigan Ann Arbor USA2University of Michigan Ann Arbor USA3University of Michigan Ann Arbor USA4University of Michigan Ann Arbor USA5Karlsruhe Institute of Technology Karlsruhe GermanyShow Abstract
The cellular microenvironment plays an integral role in cell adhesion, proliferation, and gene expression and has been shown to guide stem cell fate. Stem cells have a host of applications in tissue engineering and regenerative medicine including skeletal muscle regeneration and cardiac repair. In spite of the large therapeutic impact of stem cells, their use is hindered in part by the use of undefined substrate culture conditions which use animal-derived products leading to issues with xenogenic contamination and batch to batch variation. Some of these concerns could be eliminated through the use of synthetic substrates. Our lab has demonstrated long term maintenance of human embryonic stem cells (hESCs) on a synthetic zwitterionic polymer, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), generated using a â?ografting toâ? approach.1 However, the influence of substrate properties on cell morphology and function is not clearly understood. In order to elucidate the material properties that influence hESC behavior on PMEDSAH, a more controlled polymerization process that utilizes a â?ografting fromâ? approach, atom transfer radical polymerization (ATRP), is employed to fabricate PMEDSAH brushes. The physical properties of this polymer are modulated by brush thickness and in this work the influence of polymer chemistry on cell-substrate interactions is decoupled from the physical properties of the polymer by generating PMEDSAH brushes of varying thickness. In particular, the wettability, surface roughness, and antigen binding capabilities of the PMEDSAH brush as a function of thickness is assessed. Furthermore, the interaction of human mesenchymal stem cells (hMSCs) and hESCs on PMEDSAH coatings of varying thickness is undertaken. This work elucidates the influence of PMEDSAH material properties on hMSC and hESC adhesion, morphology, and differentiation. Results from this work may be applied to material design for stem cell culture systems. 1. Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat Biotechnol 28, 581-583 (2010).
5:30 AM - PP2.9
Nano-bottlebrush Electrospun Fibres for Investigation of Cell-substrate Interactions
Andrew E Rodda 1 2 3 David R Nisbet 4 1 Laurence Meagher 2 3 Kevin E Healy 5 John S Forsythe 1 3
1Monash University Clayton Australia2CSIRO Clayton Australia3Cooperative Research Centre for Polymers Notting Hill Australia4The Australian National University Canberra Australia5University of California Berkeley USAShow Abstract
Variation of multiple signals within a cellular microenvironment can lead to a change in cell response that is not easily predictable from investigations of single signals. Cells can respond to several types of environmental signal, including both biological signals (such as adhesion proteins, growth factors and small peptide analogs) and physical features such as topography and mechanical properties. A platform is required where both physical and biochemical signals can be varied independently of each other within a three dimensional environment. Our work has studied electrospun nanofibres produced from poly(styrene-co-vinylbenzyl chloride). The VBC monomer subunits allow for the subsequent grafting of polymer brushes onto the fibres via atom transfer radical polymerisation (ATRP) without requiring further chemical activation. This process creates a bottlebrush-like core-shell structure. Polymer brush layers containing poly(ethylene glycol) can provide a low protein fouling background, and can be co-grafted with other functional monomers. This has allowed the creation of low-fouling electrospun fibres that contain reactive groups, such as alkynes, for highly specific covalent attachment of bioactive peptides via click-chemistry. At the same time, the physical properties of the material can be altered easily through optimisation of the electrospinning and grafting conditions, independently of changes in peptide attachment. This type of material can provide a highly controlled 3D nanofibre substrate within which interfacial interactions may in the future be investigated and optimised.
5:45 AM - PP2.10
Elastic Conductive Polymer Nanofiber Scaffolds for Tissue Regeneration
Craig A. Milroy 1 Christopher Ellison 1 Christine E Schmidt 1 2
1University of Texas, Austin Austin USA2University of Texas, Austin Austin USAShow Abstract
Conducting polymers (CPs) are organic compounds with special chemical characteristics that endow them with electrical conductivity on the same order as inorganic semiconductors or metals. In addition, CPs exhibit the traditional advantages of polymeric materials: they are light, easy and inexpensive to synthesize, amenable to blending and copolymerization with other materials, and may be doped with a variety of compounds that can be adsorbed and released under desired conditions. As a result, CPs have been investigated for use in a wide range of applications such as implantable biosensors, organic solar cells, water-purification membranes, artificial muscles, advanced textiles, flexible electronics, and clinical devices for delivering biologically active compounds. The fact that a variety of cell types respond to electrical stimuli also renders CPs especially useful for tissue engineering scaffolds. However, the brittleness and poor long-term stability of CPs have greatly impeded their widespread application. To reduce the inherent brittleness of CPs, we have synthesized blends with polyurethane (PU). The improved mechanical properties of this material, compared to pure CPs, allow it to be processed into a range of morphologies (films, foams, fibers) with widely tunable mechanical and electrical properties. Once synthesized, the material may be dissolved in a variety of solvents and electrospun to produce elastic fibers with dimensions as small as 100 nanometers. The resistance of these fibers ranges from 1 â?" 20 Mohm, depending on the chemical formulation and electrospinning parameter setpoints. Although other research groups, including ours, have previously generated conductive fibers by chemically coating non-conductive nanofibers with CPs, these materials have not demonstrated appreciable elasticity. Our elastomeric fibers combine electrical conductivity, biocompatibility, and significant mechanical resilience to create a three-dimensional (3D) cell-culture microenvironment that mimics the native extracellular matrix. This hybrid scaffold thereby enables electrically-stimulated cellular growth and differentiation in a mechanically stressful environment, e.g. for peripheral nerve and cardiac tissue regeneration.
PP3: Poster Session: Manipulating Cellular Micronevirnoments
Tuesday PM, April 10, 2012
Moscone West, Level 1, Exhibit Hall
6:00 AM - PP3.1
Influence of Hydrogel Matrix Properties on Proliferation of Pancreatic beta;-cells in 3D
Asad Raza 1 Chien-Chi Lin 1
1Indiana University Purdue Univeristy Indianapolis Indianapolis USAShow Abstract
Islet transplantation is currently the only option to cure insulin-dependent (Type 1) diabetes. Unfortunately, the clinical prevalence of islet transplantation is greatly hindered by the shortage of donor islets. Thus, biomaterials capable of promoting the proliferation of insulin-secreting cells in 3D have the potential to provide large quantity of insulin secreting cell spheroids for the treatment of Type 1 diabetes. Furthermore, a strategy that allows for facile recovery of the expanded cell spheroids is beneficial for biological analysis of the expanded cell constructs. Towards achieving these goals, we utilized poly(ethylene glycol) (PEG) hydrogels formed by thiol-ene photopolymerization as a platform for 3D culture of pancreatic beta-cells (MIN6) and explored the influence of gel matrix degradation on cell proliferation and spheroid formation. We found that high macromer concentrations resulted in decreased Î²-cell survival and proliferation, which may be attributed to lower cell survival following photo-encapsulation and slower hydrolytic gel degradation over time. Macromer concentration, however, had minimum impact on the size of cell spheroids but resulted in decreased number of spheroids formed. Furthermore, we studied the influence of hydrogel degradation on the formation of cell spheroids and found that Î²-cell proliferated faster when encapsulated in gels capable of undergoing hydrolytic degradation. Using a chymotryspin-sensitive peptide linker, we further examined hydrogel erosion kinetics and its effect on recovery of naturally formed cell spheroid within the hydrogel matrix. In summary, thiol-ene hydrogels provide a cytocompatible environment for promoting the proliferation of beta-cell in 3D, and the formation and recovery of beta-cell spheroids depend largely on hydrogel properties. This hydrogel system may serve as a cell culture platform for generation and recovery of other tissue-engineered cell constructs for regenerative medicine applications.
6:00 AM - PP3.10
Fabrication of Alginate Microstrands for 3D Mimicking of the Stem Cell Microenvironment
Andrea Unser 1 Bridget Mooney 1 Christopher Bowman 1 Dennis Pu 1 Nurazhani Abdul Raof 1 Magnus Bergkvist 1 Yubing Xie 1
1University at Albany College of Nanoscale Science and Engineering Albany USAShow Abstract
The therapeutic potential of stem cells is imperative to the future of personalized medicine. This includes areas such as cell therapy, diagnostics, and drug discovery. However, in order to make use of stem cells for these purposes, it is of vital importance that their differentiation be directed into specific cell types. One component that contributes greatly to the differentiation of stem cells is their microenvironment or niche. In vivo, the microenvironment of cells is three-dimensional so it is necessary to fabricate a microenvironment of similar dimension in vitro. Another aspect of the microenvironment that must be taken into account during fabrication is its fluidity. A prominent example of a material that can take a three-dimensional shape and maintain fluidity is the hydrogel. It is proposed here that a hydrogel-microstrand microenvironment with a liquefied core will facilitate the differentiation of mouse embryonic stem cells (mESCs) into adipocytes. These microstrands were fabricated using a simple approach, which involved the dispensing of an alginate-cell solution into calcium chloride. The parameters to control the size and the swelling rate of the hydrogel microstrands were investigated. The self-assembly behaviors and growth of mESCs in alginate microstrands were studied. The cells in the microstrands were then subjected to directed differentiation into adipocytes over the course of two to three weeks. After careful execution, adipocyte differentiation within the microstrands was confirmed by oil red O staining and immunocytochemistry for adipocyte-specific markers. Samples were examined using the Nikon Eclipse 80i fluorescence microscope and Leica SP5 confocal microscope. Further characterization of these microstrands will include elasticity measurements and functional analysis of adipocytes. In summary, we fabricated three-dimensional alginate-microstrand constructs for ESC self-assembly and preferentially differentiated the mESCs into adipocytes with high efficiency. This shows significant promise in fabrication of a novel system that can direct stem cell differentiation and growth for future clinical applications.
6:00 AM - PP3.11
Tailoring Supramolecular Interactions to Control Cell Response: From Viability to Proliferation to Morphogenesis
Christina Jane Newcomb 1 Shantanu Sur 2 Samuel I Stupp 1 2 3
1Northwestern University Evanston USA2Northwestern University Chicago USA3Northwestern University Evanston USAShow Abstract
The function of a living cell relies on the balance of supramolecular forces that govern the self -assembly of molecules such as proteins and lipids. Understanding the role of these forces in a synthetic context is important for the development of extracellular mimetic materials and to elucidate events at the cell-material interface. In our approach, we utilize a platform of self-assembling peptide amphiphile (PA) molecules as a building block to form high aspect ratio nanofibers. PAs are comprised of a peptide sequence covalently linked to an alkyl tail and are modular in design, affording control over hydrophobic collapse and intermolecular hydrogen bonding, the two dominant supramolecular forces that drive their assembly. To first probe the interaction between the cell membrane and PA, we cultured our materials with MC3T3-E1 preosteoblast cells and found that assemblies with less stable hydrogen bonding induced catastrophic rupture of the cell membrane within minutes, while the nanofibers that were reinforced with ordered beta sheet hydrogen bonding or a stable hydrophobic core supported cell survival. Next, we investigated the interface between integrins and PA, and observed differences in cell adhesion and morphology by simply tailoring the supramolecular forces within the assemblies. Specifically, cells on stiffer nanofibers exhibited a flat morphology, supported by strong focal adhesion complexes and a stable actin cytoskeletal framework. In contrast, cells on softer nanofibers retained a rounded morphology with weaker focal adhesions and less organized actin filaments. Finally, we probed cell function and found that assemblies with increased hydrogen bonding promoted increased proliferation rates and alkaline phosphatase activity as compared to their weaker counterparts. Overall, our results emphasize the significance of supramolecular interactions when using artificial scaffolds in the extracellular milieu, and can provide further insight for materials design to guide biological response in a variety of applications from cancer therapy to regenerative medicine.
6:00 AM - PP3.12
Natural Polymers Based Scaffolds for Chondrogenic Differentiation of Human Mesenchymal Stem Cells
Nandita Singh 1 Sameer S Rahatekar 2 Krzysztof Koziol 3 Avinash Patil 4 Sky Ng 1 Stephen Mann 4 Anthony Hollander 1 Wael Kafienah 1
1University of Bristol Bristol United Kingdom2University of Bristol Bristol United Kingdom3University of Cambridge Cambridge United Kingdom4University of Bristol Bristol United KingdomShow Abstract
Natural polymers such as silk and chitin/chitosan have been shown to have good biocompatibility and low immune response. Unlike synthetic polymers the degraded products of these natural polymer scaffolds are not likely to damage the tissues during regeneration. We prepared scaffolds from silk and chitin/chitosan and their blends with different blend composition to test their ability to support adult mesenchymal stem cells (MSC) growth and differentiation. These scaffolds were analysed for levels of viable cell adhesion, and production of various differentiation and transcription markers using both Real time and Quantative polymerase chain reaction. Adult MSCs were grown on the pure and blended scaffolds and viability assay was done to find out surface attachment of cells followed by quantitative assay for cell attachment. Real time and quantitative PCR was conducted to determine expression for Adipogenic and Chondrogenic markers. All membranes showed good viability and proliferation of cells. No expression for adipogenic markers was observed and an up regulation of chondrogenic markers was observed. The findings suggest that these natural polymers and their blends hold a great potential to be used for MSC growth and differentiation for cartilage tissue engineering.
6:00 AM - PP3.13
Identification of Neural Crest-like Synovial Stem Cells
Fang Huang 1 Zhenyu Tang 1 Song Li 1
1University of California, Berkeley Berkeley USAShow Abstract
Recent studies suggest that bioactive scaffolds can be used to regenerate cartilage by local cell homing. However, the specific cell types participating in regeneration are not fully understood. In this study, we identified a novel type of neural crest like synovial stem cell (NCL-SSC) residing within the synovial membrane, which has several important characteristics distinct from previously identified stem cells. Protein marker screening revealed that NCL-SSCs homogeneously expressed neural crest stem cell markers such as Sox1, Sox10, Snail and Vimentin. Furthermore, differentiation assays NCL-SSCs can be induced into not only into mesenchymal lineages including osteoblasts, adipocytes, and chondrocytes, but can also differentiate into peripheral neurons and Schwann cells. In addition, we found that the combination of chick embryo extract and basic fibroblast growth factor can maintain the multipotency and maker expression of NCL-SSCs. Cloning assay showed that the average plating efficiency of NCL-SSCs is about 10% and the cloned colonies retained the marker expression and multipotency. NCL-SSCs can form neurosphere like aggregates when cultured in ultra-low attachment culture dishes. In summary, our study indentified a new type of local cells in synovial knee joint, which could be an important cell source for in situ tissue engineering.
6:00 AM - PP3.15
Direct Printing of 3D Microscaffolds for the Study of Interstitial Cell Migration
Julian Roman Schneider 1 Patrick Galliker 1 Tobias Bachmann 1 Aldo Ferrari 1 Manish K Tiwari 1 Dimos Poulikakos 1
1ETH Zurich Zurich SwitzerlandShow Abstract
We have developed a novel printing technique of colloidal inks, based on electrohydrodynamically assisted micro- and nanodroplet ejection from a microscopic nozzle. The application of an electrical potential between the ink-loaded pipette and the substrate results in the formation of a liquid meniscus and highly periodic ejection of ink droplets from its apex. The frequency of droplet formation is between 1-10 kHz and can be controlled with the voltage, while the pulse length defines the total number of deposited droplets. By careful matching of the solvent evaporation and ejection frequency, the nanoparticles in the colloidal ink can combine to form three-dimensional out-of-plane structures via self-assembly. Direct printing of dots, lines and pillars with widths well below 100 nm have been demonstrated (Galliker et al., under review, 2011). With the abovementioned technique, larger structures are also accessible simply by adopting wider nozzles and ink solvents with higher vapor pressures. By combining sequential (i.e. dotwise) and continuous printing, freeform wires and complex arc structures with diameters around 1 Âµm have been fabricated. These structures provide a controlled and precisely engineered microenvironment for the study of the migration of cells within three dimensional tissues. It has been shown that highly malignant tumor cells can plastically adapt their migration behavior to the surrounding microenvironment in order to bypass obstacles or penetrate pores (Wolf et al., J. Cell Biol. 160, 2003). The reconstituted collagen matrices mainly used for these studies on interstitial cell migration closely simulate the physiological environment, yet have limited optical accessibility and contain wide pore size variability. New platforms produced by microfabrication methods like 3D lithography (Klein et al., Adv. Mater. 23, 2011) or two-photon laser ablation (Ilina et al., Phys. Biol. 8, 2011) have recently been proposed. Until now, though, a flexible and economic method for the fabrication of large scale, mixed pore size microenvironments totally accessible by high-resolution microscopy techniques was still not available. Optically accessible cyclo-olefin-copolymer substrates were pre-structured by thermal nanoimprint lithography (NIL) to form a basal scaffold for cell contact guidance (Ferrari et al., Nano Lett. 11, 2011). Arrays of freestanding arcs with varying cross sections between 10-100 Î¼m2 were then fabricated by 3D printing of a colloidal gold nanoparticle ink. Having precise control over the pore shape, size and stiffness, the influence of these parameters on the ability of the cell to undergo interstitial pore penetration can be decoupled. The visualization of cell migration, particularly upon penetration of the pore structures is explored with cancer cells by wide-field, confocal and total internal reflection fluorescence (TIRF) microscopy.
6:00 AM - PP3.17
Cancer Adaptation to Drug Gradient in Microfluidic Microenvironments
Amy Wu 1 3 Guillaume Lambert 2 3 Robert H Austin 2 3 James C Sturm 1 3 Chira Chen-Tanyolac 4 Thea D Tlsty 4
1Princeton University Princeton USA2Princeton University Princeton USA3Princeton Institute for the Science and Technology of Materials (PRISM) Princeton USA4University of California, San Francisco San Francisco USAShow Abstract
In order to study the evolution of drug resistance in cancer, it is important to mimic the tumor microenvironment, in which cells are exposed to not uniform concentrations but rather gradients of drugs, nutrients, and other factors. However, it is hard to control the temporal and spatial profile of gradients using traditional in-vitro techniques. In this paper, we report the generation of stable gradients of doxorubicin, using a microfluidic gradient generating device for the human metastatic breast cancer cell line MDA-MB-231, and the successful long-term culture of such cells in the chip. Exposing cells in a microfluidic environment to constant fluid flow has a negative effect cell viability  (T. M. Keenan et al, Lab on a Chip, 2007). We found that flow rates as small as 100 microns/sec does change morphology of the cells after two days. We then fabricated a structure in which DC fluid flows outside the culture region are used to establish drug concentration boundary conditions, and arrays of micro-posts serve as perfusion barriers to allow the drug to diffuse across the culture chamber to establish , but isolate it from DC fluid flows. The culture of mammalian cells in microfluidic environments is much more demanding than that of bacteria such as E-Coli. MDA-MB-231 cells were introduced into the gradient zone via an extra port, and their successful on-chip culture required several factors. The chip surface was coated in-situ with appropriate extracellular matrix. A micro-incubator with controlled humidity, temperature, oxygen, and carbon dioxide concentrations and a PDMS chip sealing method was used to allow the ambient to diffuse into the culture zone were used. The input fluid for the gradient culture chip contains DMEM growth media (with 10% fetal bovine serum), and was also preconditioned with oxygen and carbon dioxide. After seeding, the breast cancer cells grow exponentially for 5 days, and then enter a stationary phase, in which the cells are still healthy with stable population after 16 days. Finally, we report the adaptation of the breast cancer cells under the stress of doxorubicin gradients (0-200nM/mm). The cell population increases quickly in the low drug region, but also increases (slower) in the high drug region. Further work is ongoing to determine whether resistance is emerging or if cells are migrating from the low to high drug concentration regions.
6:00 AM - PP3.19
Cellulose Nanowhiskers: Nanoscale Cues for Directing Skeletal Muscle Myogenesis
James M Dugan 1 Julie E Gough 1 Stephen J Eichhorn 2
1The University of Manchester Manchester United Kingdom2University of Exeter Exeter United KingdomShow Abstract
Cellulose nanowhiskers (CNWs) are high aspect-ratio nanoparticles with diameters of a few nanometres which are prepared by partial hydrolysis of native cellulose. Although normally found in plant cell walls, cellulose is also produced by some marine invertebrates. Such animal-derived cellulose is highly crystalline and allows the production of particularly high aspect-ratio CNWs with lengths on the order of a micrometre. We have shown that such CNWs are non-cytotoxic to skeletal muscle myoblasts and that their unique combination of physical, chemical and biological properties makes them a highly suitable material for engineering the cell microenvironment for applications in tissue engineering and regenerative medicine. Highly oriented surfaces of deposited CNWs were prepared using spin coating. The degree of CNW deposition and relative orientation was effectively controlled and modulated and the surfaces were characterized by atomic force microscopy and image analysis. The mean feature height was only 5.5 nm and yet the topographical cues provided by the deposited CNWs induced contact guidance in C2C12 skeletal muscle myoblasts that were seeded to the surfaces. The myoblasts adopted highly oriented morphologies and upon differentiation fused to form highly oriented arrays of myotubes. Furthermore, the degree of terminal differentiation and myoblast fusion was upregulated on the oriented CNW surfaces and the myoblasts deposited highly oriented fibrils of extracellular fibronectin. The CNW surfaces were also shown to support the adhesion of human mesenchymal stem cells (hMSCs) and to direct their morphology. Using a co-culture system the hMSCs were induced to fuse with C2C12 myotubes demonstrating that CNWs are a potentially valuable tool for engineering human skeletal muscle tissue with physiologically relevant structure across several length scales. As extracellular matrix mimics the CNWs are some of the smallest features ever demonstrated to induce contact guidance in mammalian cells.
6:00 AM - PP3.2
Engineering Intestinal Microenvironments: Progress towards a New Preclinical Drug Screening Platform
Rebecca Snyder 1 James Su 2 Calvin Kuo 3 Sarah Heilshorn 2
1Stanford University Stanford USA2Stanford University Stanford USA3Stanford University Stanford USAShow Abstract
As the cost and duration of clinical trials continue to increase, a rising emphasis is being placed on improving preclinical testing methods. The current standard preclinical absorption model utilizes human colon cancer-derived cellular monolayers grown on collagen-coated membranes. Despite its prevalence of use, this model suffers from multiple limitations, including inaccuracies in predicting paracellular transport as well as poor reproducibility. The goal of this work is to exploit the molecular-level precision of protein-engineered scaffold materials to create an improved in vitro mimic of intestinal tissue, the primary point of absorption for most orally administered drugs. We have developed a modular elastin-like protein (ELP) in which we can independently tune cell adhesive and mechanical properties. Through selectively incorporating RGD ligand domains, we can obtain up to 9300 cell adhesive ligands per square micron of ELP gel surface. Further, by altering the concentration of chemical crosslinker, we have generated thin films with elastic moduli ranging from 20 to 100 kPa. It is hypothesized that both scaffold biomechanics and biochemistry will affect cytoskeletal organization and focal adhesion formation, cell processes known to correlate with tight junction formation and therefore paracellular transport. Independent tuning of these individual properties is thus hypothesized to facilitate the use of scaffold property optimization to regulate paracellular permeability. We demonstrate that our ELP is a suitable extracellular matrix protein mimic for use as an adsorbed coating in the culture of epithelial cell lines, resulting in comparable metabolic health and cell density of confluent monolayers relative to adsorbed collagen, fibronectin, and laminin. Our results also demonstrate that this material is a suitable matrix for culturing primary intestinal organoids in 3D, enabling the maintenance of mature primary intestinal epithelial layers for up to 6 weeks in culture. We further demonstrate that replacement of the standard collagen coating with our ELP material provides a novel absorption model that performs comparably to the collagen standard, rendering statistically equivalent fluxes of a transport marker in both the apical-to-basal and basal-to-apical directions. Further scaffold property optimization will enable the generation of a new model for intestinal absorption that is not only more consistent and reliable but also facilitates the tailoring of monolayer permeability to match various conditions in which intestinal barrier function is altered.
6:00 AM - PP3.20
A Completely Synthetic, Micro-patterned Culture Surface for Human Embryonic Stem Cells
Felicia L Svedlund 1 Colleen Courtney 2 Elizabeth Irwin 1 Albert Lin 1 Kevin E Healy 1
1UC Berkeley Berkeley USA2UMBC Baltimore USAShow Abstract
Human embryonic stem cells (hESCs) have applications in numerous different areas of biological engineering, but there are still difficulties with efficiently proliferating and directing differentiation of these cells in vitro. Studying the effect of colony size and spacing on hESCs may allow for better control of their proliferation and differentiation. I have developed a soft lithography technique using PDMS stencils to micropattern an interpenetrating polymer network composed of poly(acrylamide-co-ethylene glycol). The IPN is a superior surface for blocking protein and cell adhesion because it is very robust and has been shown to reduce fibrinogen adsorption by more than 96% compared to TCPS. During patterning by oxygen plasma etching, the areas blocked by the stencil will remain non-adhesive, while the areas exposed to plasma will be etched to the base TCPS substrate. The success of this patterning has been shown by patterning 3T3 fibroblasts on this surface for up to a week. Currently, the most common surface for in vitro culture of hESCs is Matrigel, which is not well defined, contains animal products, and suffers from batch-to-batch variability. Therefore, it has been a focus of research in our lab to develop a completely defined, synthetic surface for the culture of hESCs. Work in our group has developed a low cost, synthetic polymer surface composed of aminopropylmethacrylamide (APAAm) that has been used to successfully culture hESCs for over 20 passages in mTeSR media. Immunostaining and quantitative RT-PCR studies demonstrated that cells cultured on the APAAm surface have similar proliferation and pluripotency markers to cells cultured on Matrigel. The incorporation of this material into my micro-patterned culture surface will allow for defined in vitro culture of hESCs and allow for the study of colony size and spacing on cell growth, proliferation, and differentiation.
6:00 AM - PP3.21
Characterization of a Functionalized Nanoneedle Array for Protein Detection as a High Throughput Biomarker Discovery Platform
Rahim Esfandyarpour 1 Hesaam Esfandyarpour 1 Mehdi Javanmard 1 Ronald W Davis 1
1Stanford University Stanford USAShow Abstract
Here we present the development of an array of electrical nano-biosensorsembedded in a microfluidic channel, which we hereby refer to as Nanoneedle Biosensors, capable of label-free electronic detection of protein biomarkers. The ultimate goal of the nanoneedle biosensor is to insert the needle tip into a cell membrane and make direct in-vivo measurements of protein binding inside the cell without disturbing the cell itself. In this paper, we will present the proof of concept study for protein detection. The nanoneedle biosensor is a platform, capable of electrical detection, both label free and in real-time. Our sensor, which is capable of high sensitivity detection, operates by measuring the impedance modulation across a nanoneedle tip, due to the binding of biomolecules such as proteins or nucleic acids. We show that the sensors are capable of protein detection. We functionalized the Nanoneedle Biosensors with the receptors specific to a target protein via physical adsorption for immobilization. We have used biotinylated bovine serum albumin as the receptor and different concentrations of Sterpavidin as the target analyte. The detection of Streptavidin binding to the receptor protein is presented. We also present measurements for the sensitivity of the sensor for a range of target protein concentrations.
6:00 AM - PP3.23
Photoswitchable block copolymer for micropatterning different cell types
Kyu-Shik Mun 1 Ross Andrews 1 Carlos C Co 1 Chia-Chi Ho 1
1University of Cincinnati Cincinnati USAShow Abstract
Creating micropatterned coculture of different cell types is useful for the investigation of cell behaviors and communications, or engineering tissues composed of multiple cell types. Molecules with the cell adhesive property switchable by external stimuli would provide additional capability to manipulate different cell types with more complexity. Here, we have developed a new photoresponsive molecule that can switch the cell adhesive property on the surface using a photocleavable moiety which cleaves upon exposure to long wavelength UV. We demonstrate the principle to investigate the intercellular communication between hepatocytes /endothelial cells and endothelial cells/pericytes. This method provides a tool for studying cell-cell interactions and mimicking the complex tissue structure in vitro.
6:00 AM - PP3.4
Engineering Poly(ethylene glycol) Hydrogel Microenvironments to Promote Mesenchymal Stem Cell Proliferation
Michael Hoffman 1 2 Danielle Benoit 1 2 3
1University of Rochester Rochester USA2University of Rochester Medical Center Rochester USA3University of Rochester Rochester USAShow Abstract
Although most orthopaedic fractures heal, the management of critical sized (>3mm) bone defects continues to present major challenges clinically. During normal bone healing, mesenchymal stem cells (MSCs) are recruited, proliferate, and produce extracellular matrix that subsequently becomes mineralized to form bone in a highly-orchestrated process. Therefore, a means to deliver and control MSC proliferation represents a promising regenerative strategy in bone tissue engineering. Thus, we are designing poly(ethylene glycol) (PEG) hydrogel microenvironments to exploit the action of the soluble small molecule GSK3Î² (glycogen synthase kinase 3Î²) inhibitor, 6-bromoindirubin-3â?T-oxime (BIO). Previously, we demonstrated dose dependent MSC proliferation in vitro utilizing transient (24 hr) BIO treatment. We observed a 4.5-fold and 5.5-fold increase in cell number under 2 Î¼M and 5 Î¼M BIO dosing conditions, respectively, as compared to untreated controls. In the current work, we developed a PEG-based degradable hydrogel network to provide reproducible BIO delivery and exploit BIO-mediated MSC proliferation. Various ratios of hydrolytically degradable PEG-PLA-DM [poly(lactide)-b-PEG-b-poly(lactide) dimethacrylate] and PEG-CAP-DM [poly(caprolactone)-b-PEG-b-poly(caprolactone) dimethacrylate] tri-block copolymers were tuned to degrade completely over 35 days, as required by typical bone healing. Furthermore, we utilized previously-developed controlled release strategies to tether and release BIO with tunable delivery kinetics. Total BIO dose was controlled through overall BIO conjugate incorporation, and the delivery kinetics were controlled by the number of degradable linkages incorporated into the BIO conjugate. When encapsulated in these hydrogels, MSCs exhibited high viability and similarly-enhanced proliferation as observed previously in our two-dimensional studies. We are currently exploiting these degradable, BIO-releasing hydrogel microenvironments to promote fracture healing in mouse models.
6:00 AM - PP3.5
Photo-sensitive Biocompatible Hydrogels Structuring Extracellular Environments by Two Photon Polymerisation
Jurgen Stampfl 1 Jan Torgersen 1 Aleksandr Ovsianikov 1 Xiaohua Qin 2 Zhiquan Li 2 Vladimir Mironov 1 Robert Liska 2
1TU Wien Vienna Austria2TU Wien Vienna AustriaShow Abstract
Two-Photon-Polymerisation (2PP) is a rapidly emerging platform technology for the microfabrication of three-dimensional biocompatible scaffolds for tissue engineering at the nanolevel of resolution. The required near-infrared laser emits light of minimally damaging wavelength for biological tissue. This makes it potentially very attractive to apply 2PP for developing new and advanced functional materials to be structured in direct contact with cells or other living organisms. It is crucial to design a polymerisable formulation to be minimally toxic. Furthermore toxicity assays need to be developed allowing for high throughput screening using life organisms. This paper reports the biofabrication of three-dimensional scaffolds using two-photon polymerisable hydrogels. Caenorhabditis elegans has been used as a test living organism for toxicity studies. The structuring was performed with a pulsed near-infrared laser with a wavelength of 810 nm and adjustable power up to 400 mW. Using a two-photon active, water soluble initiator (WSPI) it was possible to polymerise 3D structures in resins having M9 buffer medium contents of up to 80%; the highest aqueous content reported for the use with 2PP. High writing speeds of 10 mm/s allowed very short fabrication times. The little damaging wavelength of a near-infrared laser and a novel photo-sensitive material system with a high aqueous content made the rapid biofabrication of 3D scaffold with an embedded organism possible for the first time. These data demonstrate the feasibility and possible potential of 2PP and advanced photo-sensitive biomaterials to biofabricate 3D tissue constructs directly in the context of a living organism.
6:00 AM - PP3.6
Dual-crosslinked Alginate for Spatial Control of Hydrogel Material Properties
Julia E. Samorezov 1 Eben Alsberg 1 2
1Case Western Reserve University Cleveland USA2Case Western Reserve University Cleveland USAShow Abstract
Biomaterial properties including stiffness, degradation rate, and presentation of cell adhesion ligands are known to affect a variety of cell behaviors including spreading, proliferation, and differentiation. To engineer complex tissues, it may be important to spatially pattern these properties to create local microenvironments presenting desired signals. The capacity to achieve this in 3D would permit potential mimicry of the in vivo mechanical and chemical signaling microenvironments present during developmental or healing processes for application in tissue regeneration approaches. Here, alginate, a naturally derived polysaccharide, is used to create hydrogels with regions of different crosslinking and subsequently different material properties. Alginate can be crosslinked ionically with divalent cations, chemically with compounds such as gluteraldehyde, and in the presence of UV light and a photoinitiator if the material is appropriately modified. Methacrylated alginate, which is UV-crosslinkable, retains its ability to crosslink ionically. Because hydrogel exposure to UV light can be controlled spatially with a photomask, combining these two crosslinking mechanisms permits creation of a material that is crosslinked ionically through its bulk, and additionally crosslinked in selected areas with UV light. Alginates with 5-11% methacrylation (MA) of carboxyl groups were prepared, and for cell studies the cell adhesion peptide GGGGRGDSP was subsequently covalently attached to the methacrylated alginate. Alginates were dissolved in media containing Irgacure D-2959 photoinitiator. Calcium-only gels were crosslinked with solutions of 100 mM calcium chloride in deionized water. Dual crosslinked gels were first crosslinked with 320-500 nm light and then calcium crosslinked. In all groups, dual crosslinked hydrogels had higher shear moduli and exhibited less swelling after 48 hours than the same material crosslinked with calcium alone. These differences were statistically significant for both 7% and 11% MA hydrogels (p<0.05). Shear storage moduli ranged from 10 kPa (calcium only, 7% MA) to 25 kPa (dual crosslinking, 7% MA). Swelling ratios ranged from 40 (dual crosslinking, 11% MA) to 100 (calcium crosslinking, 7% MA). Live/dead staining demonstrated that the dual crosslinking process was not toxic to encapsulated human mesenchymal stem cells, which remained viable after one week of culture. When UV light was shone through a photomask with a 100 um checkerboard pattern onto a calcium crosslinked hydrogel, visual inspection demonstrated that this pattern was imparted to the hydrogel. Such a system will be useful for studying effects of 3D hydrogel material properties on encapsulated cells, and for patterning these properties in a scaffold for tissue regeneration applications.
6:00 AM - PP3.7
Nanotopography-based Gene Delivery and ECM Protein Patterns for Neuro-differentiation of Stem Cells
Aniruddh Solanki 1 Shreyas Shah 1 Ki-Bum Lee 1
1Rutgers University Piscataway USAShow Abstract
Neural stem cells (NSCs) are multipotent and differentiate into neurons and glial cells, which can provide essential sources of engraftable neural cells for devastating diseases such as Alzheimerâ?Ts disease, Parkinsonâ?Ts disease and spinal cord injury. One of the major challenges involved in the differentiation of NSCs is to identify and optimize factors which result in an increased proportion of NSCs differentiating into neurons as opposed to glial cells. The research toward studying the function of the two microenvironmental cues - cell-cell interactions and insoluble cues - during the neuro-differentiation of NSCs is limited, mainly due to the lack of availability of methods for the investigation. Herein, we demonstrate the effect of extracellular matrix (ECM) protein patterns and nanotopography, as insoluble cues, on the differentiation of NSCs. First, bio-surface chemistry combined with soft lithography was used to generate patterns with varying geometries and dimensions of the ECM protein, laminin, to study the influence of surface patterns and cell-cell interactions on the differentiation of NSCs. Second, we used varying nanotopographies, generated by films of different sizes of nanoparticles coated with laminin, to deliver plasmid DNA and siRNA against a specific gene to enhance the neuronal differentiation of NSCs. Our nanotopography-based gene delivery platform allows for the uptake of only the siRNA/DNA and not the nanoparticles, which is extremely advantageous. Our results confirmed that ECM protein patterns with variant geometries and dimensions guided cell-cell communications and cell-ECM interactions in a controlled manner, which ultimately led to a pattern geometry-dependent and dimension-dependent neuronal differentiation. In addition, by controlling gene expression levels using nanotopography-based delivery of siRNA, we observed a remarkably high number of NSCs undergoing neuronal differentiation. Importantly, all the experiments were carried out in the absence of exogenous factors promoting neuronal differentiation. Differentiation of NSCs into neurons was confirmed using RT-PCR and immunostaining. The down-regulation of the NSC marker (Nestin), the up-regulation of the early neuronal marker (TuJ1) and mature neuronal marker (MAP2) were monitored. Furthermore, the extent of glial differentiation of NSCs was assessed by monitoring the expression of the protein GFAP, a specific glial marker. In addition, neurite outgrowths were observed by using an inverted phase contrast microscope. Our results could be significant for tissue engineering for brain and spinal cord injuries, where NSCs can be transplanted into the damaged regions with scaffolds having specific geometries or nanotopographies for delivery of genetic material into stem cells. For example, scaffolds having nanotopographies and patterns promoting cell-cell interactions in a controlled manner could potentially lead to increased neuronal differentiation in vivo.
6:00 AM - PP3.9
Micropatterning and Covalent Immobilization of Multiple Bioactive Molecules for Regenerative Medicine Applications
Pascal Jonkheijm 1
1Mesa Institute for Nanotechnology Enschede NetherlandsShow Abstract
Incorporating bioactive molecules in biomaterials is increasingly preferred to instruct the cells and to obtain a specific and desired biological response. Surface tethered growth factors offer a better control of their spatial and temporal availability in the extracellular environment in contrast to soluble proteins. Due to challenges in simultaneous patterning of multiple biomolecules, only few studies have investigated the effect of co-patterning multiple different molecules to (stem-) cells. Such co-patterns would be very instrumental in studying the synergistic- or antagonistic activity of different bioactive molecules such as growth factors. Although inkjet printing allows arraying of multiple biomolecules, no control is available over the shape and size (>100 Âµm). Parallel patterning with micrometer level resolution and with desired shapes can be provided using microcontact printing and combined with its low fabrication costs, simplicity and reproducibility, however more wide spread implementation is challenged by the necessity of patterning m