Papers

AVS1997 Session BI-WeM: Cell Solid-Surface Interactions

Wednesday, October 22, 1997 8:40 AM in Room F

Wednesday Morning

Start	Invited?	Item
8:40 AM		BI-WeM-2 Nanopattern Transfer Using Protein Crystal Masks: A Numerical Simulation of Experimental Results T.A. Winningham, Z. Zou, K. Douglas, N.A. Clark (University of Colorado, Boulder) Ion milling has been used to transfer the nanometer-scale, periodic pattern of a protein crystal ("S-layer") into the supporting substrate.¹^,² At the same time, etch pits, attributable to the presence of surface contamination, evolve in random areas of the substrate not covered by the S-layer ("off-S-layer"). Using the theory of ripple topography proposed by Bradley and Harper,³ a computer simulation of hole formation based on curvature dependent sputtering and surface self-diffusion has been formulated. For simplicity, the study is limited to off-S-layer etch pits with the assumption that the basic mechanisms for etch pit creation are the same both on- and off-S-layer. To determine the validity of this model as applied to the patterning process, profiles of actual etch pits have been obtained using atomic force microscopy, and the simulation has been used to evolve these profiles in time. Even with a number of simplifying assumptions in place, the model simulates the experimental data quite well. ¹K. Douglas, G. Devaud, and N. Clark, Science 257, 642 (1992). ²K. Douglas, "Biomimetic Approaches to Nanostructural Fabrication," in S. Mann (Ed.), Biomimetic Materials Chemistry, VCH Publishers, New York, 1996. Chapter 5. ³M. Bradley and J. Harper, J. Vac. Sci. Technol. A 6, 2390 (1988).
9:00 AM		BI-WeM-3 Reactions of Cells to Nanotopography A. Curtis, C.D.W. Wilkinson (Glasgow & Strathclyde Universities, Scotland) Our laboratory has recently described the reactions of macrophages, endothelia, fibroblasts and epitenon cells to nanotopography. Cells were brought into contact with groove/ ridge nanotopography fabricated in silica with groove depths down to 10nm in height and with polystyrene replicas of 30nm deep pits or 30nm high cones. The groove/ridge topography led to increased adhesion, orientation, movement and elongation of the cells as well as activation of tyrosine kinases of the fyn and lyn types. The pits and cones had varying effects mainly dependent on the spacing of pits and cones. It is clear that nanofabrication provides a precise method of investigating the effects of surface roughness on cells. How do cells react to structures of the same height as protein molecules? Two hypotheses will be put forward. The first one is that cells are actively stretching on the topography and that stretch receptors are activated, which in turn activates signalling systems in the cell. The second hypothesis is that gene expression is altered by distortion of the nucleus, which may be as much as a sevenfold increase in length and a corresponding narrowing. Evidence for each hypothesis will be presented.
9:40 AM		BI-WeM-5 Fabrication and Analysis of Patterned Neuronal Networks J.J. Hickman, M.S. Ravenscroft, K.E. Bateman, K.M. Shaffer, J.J. Pancrazio (Science Applications International Corp.); Q.Y. Liu, J.L. Barker, A.E. Schaffner (National Institute of Neurological Disorders and Stroke) Information processing by neuronal networks is dependent on the intrinsic biophysical properties of the neuronal elements as well as the strength and inhibitory or excitatory nature of the synaptic connections. We are exploring the utility of neuronal patterning as a reductionist means of understanding neuronal network processing. High resolution deep UV excimer laser lithography (193 nm) has been employed to fabricate circuit patterns consisting of cell permissive and repulsive self-assembled monolayers; the resulting pattern induces neuronal adhesion and promotes neurite outgrowth in a geometrically controlled manner. Hippocampal neurons were dissociated from embryonic 19 rate in serum-free defined media at a density of 115-230 cells/mm² depending on the pattern. Morphological assessment of cell adhesion and process development was monitored using a time-lapse system. We also conducted parallel experiments using the dual patch-clamp technique to examine the functional development of both spontaneous and evoked neurotransmission in the fabricated neuronal circuit. Protonation of the primary amine of DETA significantly enhanced pattern fidelity and appeared to have a trophic effect on neurite extension, consistent with previous work (Stenger et al., 1993, Brain Res. 630: 136). After day 7 in culture, both spontaneous and evoked GABAergic neurotransmission was observed on the control unpatterned cell permissive surface, however only spontaneous release was detected on the patterned substrate. Consequences of these results for neuronal development will be discussed. This work supported by the Department of Energy and the Office of Naval Research.
10:00 AM		BI-WeM-6 Central Nervous System Cell Attachment on Chemically and Topographically Patterned Surfaces S. Turner (Cornell University); L. Kam (Rensselaer Polytechnic Institute); R. Davis, H.G. Craighead, M. Isaacson (Cornell University); J. Turner, W. Shain (School of Public Health) We are investigating the microscopic morphological and chemical cues that influence central nervous system (CNS) cell attachment to silicon substrates. Several different masking and reactive ion etching processes were used to selectively pattern 10-100 nm scale surface texture on Si. Photolithography was used to pattern the textured areas, presenting side-by-side regions of varying surface character to cells in cultures. Cells were then fixed and stained for examination. Cell attachment and morphology were observed by laser scanning confocal fluorescence microscopy and scanning electron microscopy SEM. Interestingly, the nature of the response was different for the two types of cells used in this study. Cells from a continuous cell line (LRM55) seemed to avoid attachment with the rougher surface, favoring the smoother secondary surface. In contrast, the primary rat cortical astrocytes showed a preference for rougher surfaces. We are also using electron beam lithography to create a range of well characterized surface features with dimensions down to ~40 nm. We have previously examined the effects of several surface chemical treatments on smooth Si substrates and we are now combining the chemical treatments with surface topography to determine which may dominate in certain cases. Along with methods for changing surface chemistry, the application of patterned surface texture provides us additional flexibility in guiding the growth and attachment of CNS cells on inorganic surfaces. This will have impact on biological implant devices and research on nervous system function.
10:20 AM		BI-WeM-7 Modulation of Aminal Cells on Potential Controlled Electrode. M. Aizawa (Tokyo Institute of Technology, Japan) Cellular proliferation rates of various animal cells were found to be dependent on electrode potential , when they were cultured on the electrode surface. Most of cells stop proliferating in the potential range around +0.4 V vs. Ag/AgCl. The electrically regulated cell culture has been proposed, where the proliferation rate is controlled by adjusting the electrode potential. A possible mechanism is discussed. PC12 cells proliferate under normal culture medium even on the electrode surface, but stop proliferating and differentiate to grow neurites by a stimulation of nerve growth factor (NGF). We have succeeded, however, in initiating PC12 cells to differentiate in the absence of NGF by a small alternative potential of electric stimulation. The cells were cultured on the electrode surface, and proliferated as normal when the electrode was free from potential application. In contrast, the cells started outgrowing neurites in a similar manner as the NGF-stimulated differentiation when they were stimulated by a 200 mV of rectangular wave at 100 Hz. These findings suggest a possibility to initiate differentiation of other animal cells by electric stimulation. Electrically stimulated differentiation of bone narrow cells and others will be presented [1]. There was found another electric effect on astroglial cells in such a way that a gene expression process could be activated to generate and excrete NGF [2]. A sine wave of potential at 30-100 Hz was effective on such a protein production. The electric effects on each step of the gene expression pathway will be presented and the mechanism will be discussed. [1] S. Koyama, T. Haruyama, E. Kobatake, and M. Aizawa, Nature Biotechnology, 15, 164-166 (1997). [2] M. Mie, H. Ohgushi, T. Haruyama, E. Kobatake, and M. Aizawa, Cell. Eng., 1, 153-158 (1997).
11:00 AM		BI-WeM-9 Electrical Recordings from Electrogenic Cells using a Field-Effect Transistor Array. A. Offenäusser, C. Sprössler, W. Knoll (Max-Planck-Institute for Polymer Research, Germany) Exploration of the development and plasticity of electrical interactions among single cells would be greatly facilitated by a convenient non-destructive method for maintaining electrical contact with an individual culture, at a large number of recording sites, over periods of days or weeks. Field-effect transistor (FET) arrays ¹ were used to record signals from electrically active cells. The first cell system we have started to study are hippocampal neurons which exhibit several features of interest for our approach. To promote the growth and survival of these neurons the presence of nerve growth factors are necessary. In order to substitute the neurotrophic substances we have used laminin peptides covalently attached to the silicon oxide surface of the transistor array. This method allowed us to grow hippocampal neurons on defined surfaces of organic thin films in chemically defined media ². Using this approach passive electrical membrane signals of hippocampal neurons were monitored with our FET-array. The second cell system we will discuss are rat cardiac muscle cells cultured on the device surface. Electrical signals from these cells were recorded with the FET’s after several days in culture. The signals can be distinguished into two types: a first type which matches in shape the intracellular signals and which can be up to 25 mV in amplitude and a second type which corresponds to the first derivative of the intracellular signals and which is less then 1 mV in amplitude. Such cell - FET assemblies may provide a means for studying the roles of various factors on the electrical activity of those cells, and have potential application in pharmaceutical research. ¹A. Offenhausser, C. Sprossler, M. Matsuzawa, W. Knoll: Field-Effect Transistor Array for Monitoring Electrical Activity from Mammalian Neurons in Culture. Biosensors&Bioelectronics (in press) ²A. Offenhausser, C. Sprossler, M. Matsuzawa, W. Knoll: Electrophysiological development of embryonic hippocampal neurons from the rat grown on synthetic thin films. Neurosci. Lett. 223, 9-12 (1997).
11:20 AM		BI-WeM-10 Diffractive Cell Detection using a Microcontact Printed Antibody Grating P.M. St. John, N. Cady, C.A. Batt, H.G. Craighead (Cornell University) We have fabricated an optical detector that is specific for targeted cells by stamping an antibody grating pattern on a silicon substrate. The antibody grating alone produces insignificant optical diffraction, but with the binding of cells, the optical phase change produces a pronounced diffraction pattern. This technique eliminates much of the surface modification and the secondary immunochemical or enzyme-linked steps that are common in immunoassays. Microcontact printing¹ provides an alternative to previously reported photolithographic-mediated techniques.² We have stamped antibodies onto clean oxidized silicon substrates with no other chemical surface treatments. Direct binding of the these antibodies to the oxidized silicon occurs in a way that still allows the antibodies to function and selectively bind antigen. The performance of the sensor was evaluated by binding Escherichia coli O157:H7 cells to the appropriate antibody stamped grating lines and measuring the diffraction intensity of a HeNe laser beam at different cell coverage densities. ¹A. Kumar and G. M. Whitesides, Appl. Phys. Lett.63, 4 (1993). ²Y. G. Tsay et al. Clin. Chem. 37, 1502 (1991).
11:40 AM		BI-WeM-11 Cell-Based Sensor Microelectrode Array Characterized by XPS, SEM, and Impedance Spectroscopy. D.R. Jung, D. Cuttino, J.J. Pancrazio, M. Czarnaski (Science Applications International Corp.); G.T.A. Kovacs (Stanford University); D.A. Stenger (Naval Research Laboratory); J.J. Hickman (Science Applications International Corp.) We are using living neurons as sensor elements for toxin detection. The cells act as both the sensing element and as the transducer, and the microelectrodes as the signal pickup. Electroplating each 14 micron-diameter electrode with Pt lowers the electrical impedance and allows efficient coupling of the cellular action potential to the electronic amplifier of the sensor. A 32-pad Au microelectrode array is characterized by XPS, SEM, and impedance spectroscopy before and after electroplating with Pt. We present SEM images of the morphology of the Pt deposits and the corresponding electrode impedances. For detection of thin Pt deposits not found by SEM, two modes of spatially-mapped XPS are compared and contrasted: high resolution imaging (2 micron) and small-area analysis (60 micron) with sample-stage stepping. Well-plated electrodes showed reductions in the real part of the impedance from 10⁸ ohms or more to 10⁶ ohms at 100 Hz, as measured using a frequency-dependent lock-in amplifier technique. Equivalent circuit models for the impedance test circuit and for the coupling of the neuronal action potential to the electrode/amplifier system are discussed. Recordings of biopotentials from neurons on platinized and non-platinized electrodes are presented.