Abstract
A microstamping technique has been developed for high-resolution patterning of proteins on glass substrates for the localisation of neurons and their axons and dendrites. The patterning process uses a microfabricated polydimethylsiloxane stamp with micrometer length features to transfer multiple types of biomolecules to silanederivatised substrates, using glutaraldehyde as a homobifunctional linker. To test the efficacy of the procedure, substrates are compared in which poly-d-lysine (PDL) was physisorbed and patterned by photoresist with those stamped with PDL. Fluorescein isothiocyanate labelled poly-I-lysine was used to verify the presence and uniformity of the patterns on the glass substrates. As a biological assay, B104 neuroblastoma cells were plated on stamped and physisorbed glass coverslips. Pattern compliance was determined as the percentage of cells on the pattern 8h after plating. Results indicate that the stamping and photoresist patterning procedure are equivalent. Substrates stamped with PDL had an average pattern compliance of 52.6±4.4%, compared to 54.6±8.1% for physisorbed substrates. Measures of background avoidance were also equivalent. As the procedure permits successive stamping of multiple proteins, each with its own micropattern, it should be very useful for defining complex substrates to assist in cell patterning and other cell guidance studies.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Britland, S., Perridge, C., Denyer, M., Curtis, A. andWilkinson, C. (1996): ‘Morphogenetic guidance cues can interact synergestically and hierarchically in steering nerve cell growth,’Exp. Biol. Online,1, (2)
Carter, S. B. (1965): ‘Principles of cell motility. The directionality of cell movement and cancer invasion,’Nature,208, pp. 1183–1187
Corey, J. M., Brunette, A. L., Chen, M. S., Weyhenmeyer, J. A. andWheeler, B. C. (1997): ‘Differentiated B104 neuroblastoma cells are a high-resolution assay for micropatterned substrates,’J. Neurosci. Meth.,75, pp. 91–97
Corey, J. M., Wheeler, B. C. andBrewer, G. J. (1991a): ‘Compliance of hippocampal neurons to patterned substrate networks,’J. Neurosci. Res.,30, pp. 300–307
Corey, J. M., Wheeler, B. C. andBrewer, G. J. (1991b): ‘Localization of hippocampal neurons to chemically modified siliton nitride surfaces.’Ann. Meeting of the Society for Neuroscience. Vol. 17, 210, New Orleans, USA
Corey, J. M., Wheeler, B. C. andBrewer, G. J. (1996): ‘Micrometer resolution silane-based patterning of hippocampal neurons: Critical variables in photoresist and laser ablation processes for substrate fabrication,’IEEE Trans.,BME-43, pp. 944–954
Curtis, A. S. G. andClark, P. (1990): ‘The effects of topographic and mechanical properties of materials on cell behaviour,’Crit. Rev. Biocomput.,5, pp. 343–362
Dulcey, C., Gregor, J., Krauthamer, V., Stenger, D., Fare, T. andCalvert, J. (1991): ‘Deep UV photochemistry of chemisorbed monolayers: Patterned coplanar molecular assemblies,’Science,252, pp. 551–554
Elender, G., Khner, M. andSackmann, E. (1996): ‘Funtionalisation of Si/SiO2 and glass surfaces with ultrathin dextran films and deposition of lipid bilayers,’Biosens. Bioelectron.,11, pp. 565–577
Goodman, C. S. andShatz, C. J. (1993): ‘Developmental mechanisms that generate precise patterns of neuronal connectivity,’Cell,72/Neuron,10,Suppl., pp. 77–98
Hammarback, J. A., Palm, S. L., Furcht, L. T. andLetourneau, P. C. (1985): ‘Guidance of neurite outgrowth by pathways of substratum-adsorbed laminin,’J. Neurosci. Res.,13, pp. 213–220
Hickman, J. J., Bhatia, S. K., Quong, J. N., Shoen, P., Stenger, D. A., Pike, C. andCotman, C. W. (1994): ‘Rational pattern design for in vitro cellular networks using surface photochemistry,’J. Vac. Sci. Technol. A,12, pp. 607–616
Ivanova, O. Y. andMargolis, L. B. (1973): ‘The use of phospholipid film for shaping cell cultures,’Nature,242, pp. 200–201
Jeon, N. L., Clem, P. G., Nuzzo, R. G. andPayne, D. A. (1995): ‘Patterning of dielectric oxide thin layers by microcontact printing of self-assembled monolayers,’J. Mat. Res.,10, pp. 2996–2999
Kendall, D. L. (1975): ‘On etching very narrow grooves in silicon,’Appl. Phys. Lett.,26, pp. 195–198
Kleinfeld, D., Kahler, K. H. andHockberger, P. E. (1988): ‘Controlled outgrowth of dissociated neurons on patterned substrates,’J. Neurosci.,8, pp. 4098–4120
Letourneau, P. (1975): ‘Cell-to-substratum adhesion and guidance of axonal elongation,’Dev. Biol.,44, pp. 92–101
Lom, B., Healy, K. E. andHockberger, P. E. (1993): ‘A versatile technique for patterning biomolecules onto glass substrates,’J. Neurosci. Meth.,50, pp. 385–397
Lopez, G. P., Albers, M. W., Schreiber, S. L., Carroll, R., Peralta, E. andWhitesides, G. M. (1993): ‘Convenient methods for patterning the adhesion of mammalian cells to surfaces using self-assembled monolayers of aklanethiolates on gold,’J. Am. Chem. Soc.,115, pp. 5877–5878
Matsuzawa, M., Liesi, P. andKnoll, W. (1996): ‘Chemically modifying glass surfaces to study substratum-guided neurite outgrowth in culture,’J. Neurosci. Meth.,69, pp. 189–196
Nelson, P. G., Yu, C., Fields, R. D. andNeale, E. A. (1989): ‘Synaptic connections in vitro: modulation of number and efficacy by electrical activity,’Science,244, pp. 585–587
Schubert, D., Brass, B. andDumas, J. P. (1986): ‘Protein complexity of central nervous system cell lines,’J. Neurosci.,6, pp. 2829–2836
Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J. H., Culp, W. andBrandt, B. L. (1974): ‘Clonal cell lines from the rate central nervous system,’Nature,249, pp. 224–227
Singhvi, R., Kumar, A., Lopez, G. P., Stephanopolous, G. N., Wang, D. I. C., Whitesides, G. M. andIngber, D. E. (1994): ‘Engineering cell shape and function,’Science,264, pp. 696–698
Stenger, D. A., Georger, J. H., Dulcey, C. S., Hickman, J. J., Rudolph, A. S., Nielsen, T. B., McCort, S. M. andCalvert, J. M. (1992): ‘Coplanar molecular assemblies of amino- and perfluorinates alkylsilanes: characterizatiopn and geometric definition of mammalian cell adhesion and growth,’J. Am. Chem. Soc.,114, pp. 8435–8442
Uenishi, Y., Tsugai, M. andMehregany, M. (1995): ‘Micro-optomechanical devices fabricated by anisotropic etching of (110) silicon,’J. Micromech. Microeng.,5, pp. 305–312
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Branch, D.W., Corey, J.M., Weyhenmeyer, J.A. et al. Microstamp patterns of biomolecules for high-resolution neuronal networks. Med. Biol. Eng. Comput. 36, 135–141 (1998). https://doi.org/10.1007/BF02522871
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02522871