AVS2001 Session BI-ThA: Cell-Surface Interaction

Thursday, November 1, 2001 2:40 PM in Room 102

Thursday Afternoon

Start	Invited?	Item
2:40 PM		BI-ThA-3 Developmental Studies of Electrical Activity of Artificially Constructed Neuronal Cell Networks C.D. James, A.J. Spence, H.G. Craighead, M.S. Isaacson (Cornell University); N. Dowell, W. Shain, J. Turner (Wadsworth Center) The hippocampus has been implicated in a range of brain functions such as the internal representation of space and memory consolidation. Dissociated hippocampal pyramidal cell cultures have yielded vital information about single unit and small network electrophysiology, yet monitoring synaptogenesis and the development of electrical activity within cell networks has proved to be a difficult task. The construction of neuronal cell networks has been investigated by many researchers for this purpose, and our labs have utilized microcontact printing and microfabricated electrode arrays to construct and study cell networks. Selective spatial organization of proteins and molecules have been used to direct neuronal cell attachment and neurite outgrowth in vitro, while microelectrode arrays allow long-term, non-invasive studies on developing network populations. The combination of both technologies has allowed our labs to monitor field and action potentials of designed cell networks in order to investigate the relevance of such factors as cell morphology and neuron-substrate interaction on the development and stability of connected units. Multi- and single-unit extracellular potentials of 50 to 300 microvolts have been observed and recorded with five simultaneous channels to enable single unit discrimination. Whole cell recordings were also performed to provide guidance in isolating single units in our extracellular recordings, while immunochemical staining of networks for synaptic proteins such as synaptophysin and PSD-95 was used to identify putative synapses. We believe that such studies may be able to provide valuable information about the maturation of coordinated activity between cells, primarily in regards to the influence of neuron morphology on action potential invasion into the somato-dendritic regions of firing cells, as well as on the developmental segregation and distribution within cells of relevant molecules such as ion channels and synaptic proteins.
3:00 PM		BI-ThA-4 The Affects of Geometric Constraints on Neuronal Process Extension A.M.P. Turner, S.W.P. Turner, R. Terao, H.G. Craighead (Cornell University); N. Dowell, W. Shain (NYS DOH Wadsworth Center); G. Withers, G. Banker (Oregon Health Sciences University) Our research has involved the study of how central nervous system (CNS) cells attach to and grow on surfaces topographically modified with micrometer-sized features. In particular, we have studied the growth of rat hippocampal neurons on surfaces patterned with pillars. Patterned silicon substrates were made using conventional semiconductor methods and polymer embossing techniques were used to make transparent substrates. It was observed that the geometric constraints to which a neuron is exposed have a significant impact on various aspects of neuronal process development, including the rate of neurite (dendritic and axonal) outgrowth, neurite morphology, dendritic branching, and specific protein production, transport and organization. Fluorescence, scanning electron, and phase-contrast time-lapse microscopies were used to analyze and quantify the growth of neurons on surfaces with 1 to 2 µm tall pillars of various widths, 500 nm to 2 µm, and inter-pillar spacings, 1.0 µm to 5 µm. We observed a 50 percent increase in the rate of neurite outgrowth on surfaces with pillars versus smooth surfaces. It was also observed that in arrays with spacings less than 2 µm, the majority of neurites grow along 90 and 45 degree paths from the soma whereas with spacings of 4 µm and greater, neurites revert back to morphologies observed on smooth surfaces. Dendritic branching was found to increase with a decrease in inter-pillar spacing and immunochemical staining demonstrated various correlations between protein organization and pillar locations. The goal of these studies is to learn more about the fundamental interactions between CNS cells and surface structure.
3:20 PM		BI-ThA-5 The Use of Surface Composition to Control Cell Phenotype Expression J.J. Hickman, P. Molnar, G. Jacob, M. Das, T. Tauber (Clemson University) There is currently a large amount of interest in neuronal STEM cell manipulation to create stable phenotypes. The initial phase of CNS development is characterized by the proliferation of the precursor cells, followed by the generation of neurons and glia. The neurons are differentiated into different neurotransmitter phenotypes as well as glial cells. However, the factors that control the differentiation of the precursor cells into differentiated cell types are still mainly unknown. It is believed that the cell environment plays a key role in the specification of neuronal cells, even though a cell intrinsic developmental program is important in regulating cell lineage. We have shown it may be possible to manipulate the development of specific phenotypes through cell-surface interactions. In the present study, the expression of neuronal cell phenotype was examined in a defined in vitro system in which embryonic rat cortical cells were grown on silica substrates modified with artificial surfaces composed of silane self-assembled monolayers (SAMs) in serum-free medium. Experiments were conducted utilizing various neurotrophic factors and various substrates to examine cortical neuron phenotype expression. Cultures were immunostained with a panel of antibodies to detect specific differentiation markers. On poly-D-lysine and DETA, glutamatergic cells represented 30-40% of total cells and GABAergic cells represented about 50-60% of total cells, which is consistent with immunocytochemical findings in vivo. On 13F the ratio of glutamatergic to GABAergic was greater. We will present these results as well as an explaination for the observed effects.
3:40 PM		BI-ThA-6 Stretching and Fibroblast Growth on GRGDSP-Peptide Modified Silicone Membranes L. Hanley, S.S. Lateef, S. Boateng, T.J. Hartman, C. Crot, B. Russell (University of Illinois at Chicago) Diseased, hypertrophic human heart muscle cells (cardiac myocytes) are found to increase in length and volume due to excessive mechanical load. We are developing an entirely new cell culture in silicone elastomer that will mimic the in-vivo cell phenotype to address such questions in cardiac mechanobiology. We chemically modify silicone membranes to improve their ability to culture cardiac myocytes under dynamic stretching, thereby allowing study of mechanical effects. It is well known from studies of cellular attachment that several intrinsic proteins found on the cell surface will recognize and attach to the GRGDSP peptide sequence. We plasma oxidize the surface of the silicone membrane; functionalize it with amine via reaction with 3-aminopropyltriethoxysilane; attach a sulfo-maleimide cross-linker; then attach a 15-residue peptide, acetyl-CGGEGYGEGRGDSPG-amide, to the cross-linker through its terminal thiol group. The membranes are characterized by x-ray photoelectron spectroscopy, spectochemical analysis, and radiolabelling. Stretching studies with radiolabelled cysteine (in place of peptide) show that the modified layer survives on the surface for 48 hours of stretching in cell culture media. The GRGDSP peptide bound silicone shows enhanced binding of rat fibroblasts when compared with amine-functionalized and unmodified silicone surfaces.
4:00 PM		BI-ThA-7 Surface Characterization and HCAEC Adhesion Studies of IPN Modified 316 L Stainless Steel G.M. Harbers (Northwestern University); T.A. Barber, M.E. Yanez, H.B. Larman, K.E. Healy (University of California, Berkeley) Interactions between synthetic biomaterials and components of the cardiovascular system still remain poorly understood. In particular, the process of restenosis following intravascular stent deployment remains a significant problem. Coatings that minimize protein adsorption and monocyte adhesion and proliferation may reduce late-term in-stent restenosis and prevent secondary interventions. In this work, a previously developed non-fouling P(AAm-co-EG/AA) interpenetrating polymer network (IPN) was applied to clinically relevant cardiovascular stent material (316L SS). The transfer of the technology from previous substrates (quartz, TiO₂/Ti, polystyrene) to SS was confirmed using water contact angle goniometry, XPS, and cell-material interactions. Water contact angle data was similar to what was previously reported for quartz substrates and XPS confirmed the addition of each subsequent layer. To test the ability of the modified material to resist cell adhesion, substrates were seeded with primary human coronary artery endothelial cells (HCAECs). Following a 24h incubation, cells were labeled and examined using fluorescent microscopy. HCAECs adhered to both the unmodified SS and the positive control (TCPS) but not to the IPN modified material (TCPS>SS>>IPN~PEG(NH₂)₂; 8875±2128, 6972±721, 124±22, and 99±29 cells/cm² on respective surfaces). Cells on unmodified SS coupons had a similar morphology to those seeded onto TCPS. However, the few viable cells that attached to the IPN and PEG(NH₂)₂ remained spherical and non-spread. It has been proposed that endothelialization of the stent surface can improve stent performance by creating a native tissue layer. Therefore, since the IPN/316L SS system is amenable to peptide modification, the identification of endothelial cell specific peptides to promote preferential endothelial cell adhesion, migration, and proliferation is under investigation.
4:20 PM		BI-ThA-8 Improved Functionalization for Chemically Patterned Polystyrene Surfaces A.A. Meyer-Plath, K. Schröder, A. Ohl (Institute of Non-thermal Plasma Physics, Germany) Tight contact between living cells and polymeric materials is a key characteristic of implant materials, medical, pharmaceutical diagnostic devices, and in vitro cell culturing. To improve cell adhesion and growth, surface modification is required for almost all types of polymer. Plasma-chemically introduced functional groups are widely used for this purpose. Type and density of surface functionalities control adsorption behaviour of cell-signaling molecules. Also, selective immobilization of biologically active molecules (e.g. attachment factors) is possible. This way, the polymer surface provokes cell responses. Chemical patterns for different cellular responses are the basis for some advanced applications of biomaterials. They may directly induce selective cell adhesion. Plasma functionalization is the basis for pattern generation. Here, continuous wave and pulsed microwave and radio frequency plasmas in nitrogen-containing gas mixtures were studied for grafting of nitrogen functional groups on polystyrene. Plasma conditions were optimized in two respects: either to obtain a high selectivity for amino groups, or to maximize the overall density of nitrogen groups. The obtained functionalized surfaces were investigated by means of XPS, contact angles and cell culture. Specific plasma conditions lead to surfaces with high-density cultures of adherent cells after 24 hours of culturing, exceeding significantly densities on the untreated or oxygen-plasma-treated polymer. The highest level of nitrogen and amino functionalization was obtained using pulsed microwave plasmas. Patterning of the chemical functionalization was realized by a hydrogen plasma treatment using a laser-cut metal mask. The chemical pattern was verified by XPS with high local resolution.
4:40 PM		BI-ThA-9 Controlled Cell Adhesion on Honeycomb Films of Biodegradable Polymers T. Nishikawa (RIKEN Frontier Research System, Japan); K. Nishikawa, R. Ookura, J. Nishida (Hokkaido University, Japan); K. Arai, J. Hayashi (RIKEN Frontier Research System, Japan); M. Matsushita, S. Todo (Hokkaido University, Japan); M. Hara, M. Shimomura (RIKEN Frontier Research System, Japan) We report that a honeycomb like micro-porous film (honeycomb film) can control cell adhesion of hepatocytes and cardiac myocytes. The honeycomb films were fabricated by casting a dilute solution containing biodegradable polymers (poly-L-lactic acid (PLLA) and poly-ε-caprolactone (PCL)) and an amphiphilic polymer on water surface in a humid atmosphere. By the method, self-supported honeycomb films were obtained. Hepatocytes were cultured on a self-supported honeycomb film of PLLA. The cells formed a single layer of columnar shape cells with a thickness of 20 µm. The tissue formation of hepatocytes specifically occurred on the honeycomb films of PLLA, but not on flat films of PLLA. The artificial tissue of hepatocyets expressed high level of albumin secretion, which was comparable to that of spheroids of hepatocytes. Furthermore we succeeded in three dimensional culturing of hepatocytes. Hepatocytes formed two single layers on each sides of a self-supported honeycomb film of PLLA. Honeycomb film of PCL was stretched out uniaxially by mechanical force. The honeycomb pores were deformed into elongated hexagons and rectangles. Since the array of the elongated hexagons is anisotropic, the stretched honeycomb film is applicable to guiding cell alignment. We used a stretched honeycomb film of PCL as a cell culture substrate for cardiac myocytes. The substrate was fabricated by placing a stretched honeycomb film of PCL onto a glass plate. Cardiac myocytes of rat embryo were not aligned in a specific direction on regular honeycomb patterned surface. On the other hand, cardiac myocytes were aligned along the long axis of the stretched micro-pores on the stretched honeycomb film. Thus the honeycomb films can control cell alignment as well as cell attachment. Based on the results, we expect that the honeycomb films can be designed, fabricated, and utilized in accordance with target tissues.
5:00 PM		BI-ThA-10 New Substrates for Retinal Cell Transplantation C.J. Lee, S.F. Bent, P. Huie, M. Blumenkranz, H. Fishman (Stanford University) A novel treatment for age related macular degeneration (AMD) is currently being investigated. This treatment involves the transplantation of human pigment epithelial cells (PE) on a carrier substrate to rescue the diseased retina. Various substrates including synthetic biodegradable polymers and biocompatible substances have been proposed as carrier substrates. Biocompatible materials offer the ability to coexist in the subretinal space, thus reducing immune rejection. The goal of this work was to grow cells on various biocompatible materials and to show the survival and longevity of the cells. Without specific constraints, the cells exhibit a variety of morphologies, including cuboidal and elliptical structures. In this study, surface modifications were employed to control the growth and morphologies of the cells. The cells have been successfully grown on these modified substrates, exhibiting stable function for at least two weeks in culture. In summary, we show that engineering biocompatible substrates is possible and that stable growth of cells occurs. The possibility of the feasibility of this treatment in animals will be discussed.