RPE1 cells seeded on fibronectin patterns were constrained for days on the .... cell internal organization and oentation of polarity,PNAS, pp. 19771â19776,.
A SIMPLE AND VERSATILE METHOD FOR SINGLE CELL PATTERNING Ammar Azioune1, Manuel Théry2, Michel Bornens1 and Matthieu Piel1 1
2
Institut Curie, UMR 144, Paris, FRANCE Commissariat à l’Energie Atomique (CEA), Grenoble, FRANCE
ABSTRACT This work describes a simple and versatile method for protein patterning on surfaces covered with polyethylene glycol (PEG) hydrogel. Pegylated glass and polystyrene surfaces were exposed to deep UV light through a synthetic photomask. Incubation of the proteins on the irradiated surface, resulted in well defined patterns, with high resolution. RPE1 cells seeded on fibronectin patterns were constrained for days on the PEG-coated glass, and for weeks on the polystyrene patterned surfaces. Moreover, the patterned substrates, in the absence of proteins, are stable for months in the lab atmosphere, which makes this method an alternative for academic and industrial applications. KEYWORDS: Deep UV, Antifouling, PEG, Micropatterning, Cell biology. INTRODUCTION Bio-micropatterning is an elegant method to control cell adhesion geometry on surfaces. Recently, the technique was used to address biological questions like adhesive control of cell division axis, cell internal organization, cell migration and cell polarity [1-3]. The most popular techniques used for bio-micro-patterning on flat surfaces are well documented and discussed in the literature [4-6]. Here we propose the simplest robust method, to our knowledge, to directly pattern an optimal antifouling substrate and produce protein binding regions in an inert background that fully prevents protein adsorption. We present examples of protein and cell patterning on PEG-coated glass coverslips, and on polystyrene substrates using deep UV. The resulting surfaces contain well defined adhesive (UV exposed) and non-adhesive (UV masked) regions. The UV exposed regions exhibit high content of reactive chemical groups, which allow the covalent binding of proteins, and consequently long term cell patterns stability. More importantly, the patterned surfaces are stable for months in the lab atmosphere. EXPERIMENTAL Cleaned and UV activated glass coverslips were incubated with 1% m-PEG silane, or Cl-PEG silane (Gelest) overnight in ethanol absolute, rinsed twice with the same solvent, sonicated during 15 min, dried and heated for 1h at 60°C. Coating the glass surfaces with PLL-g-PEG is described in [6]. Non treated polystyrene dishes were used as received, or activated and treated like glass coverslips for pegylation. Pegylated substrates (or plain polystyrene plates) were placed on a chromium synthetic quartz photomask (Delta mask, Netherlands) with a drop of water . The mask and the substrate were placed under deep UV light (Heraeus, 4 X 60W) for 5 min.
Prior to adding cells, the patterned substrates were incubated with a mix of fibronectin (Sigma)/fibrinogen-Alexa fluor 488 nm (Invitrogen), at resp. 25 and 10 µg/ml in NaHCO3 buffer, pH 8.5 and RT. In the case of plain polystyrene, the patterned surface was incubated with pluronic (0.2 % in PBS), prior to proteins incubation. RESULTS AND DISCUSSION Figure 1 shows images of patterned fibronectin/fibrinogen-Alexa fluoro 488 on PEG coated-glass surfaces. The coated coverslips display a homogeneous immobilization of fibronectin/fibrinogen-Alexa 488 in the deep UV exposed regions, with a very good contrast showing the efficiency of the PEG coating. Fibronectin/fibrinogen patterned on PLL-g-PEG can be kept in PBS at 4°C for a few weeks and retain a good cell patterning capacity. Individual RPE1 cells precisely adopt the shape imposed by fibronectin/fibrinogen patterns and can be constrained for more than 10 days on the patterned glass substrates, and for weeks on the patterned PS. RPE1 cells are also nicely patterned on PEG-silane coated – glass surfaces, however, after 24h, cells could escape from the patterns and spread over the non UV exposed regions. This might be due to the short PEG chains, which are only 6-9 repeat units in our experiments (much less than 45 - 47 PEG monomers of the PLL-g-PEG and of the m-PEG-urea, which were found to be optimal for reducing the unspecific protein adsorption and the number of adherent cells [7, 8]). Since long PEG silanes are not commercially available, the achievement of glass pegylation with long PEG chains can only be carried out in two steps, which is time consuming [8]. For most studies on patterned cells, a confinement of cells for a few days is sufficient, making the glass covered with PLL-g-PEG by electrostatic adsorption the simplest and the faster procedure to produce protein and cell antifouling surfaces that can be directly patterned by deep UV irradiation.
Figure 1. Patterned glass coverslips covered with: (a) m-PEG silane, (b) ClPEG silane, (c) PLL-g-PEG, after 4h of incubation with RP1 cells in DMEM-F12, 10%FCS. After 24h, RPE1 cells escapde the protein patternes on the m-PEG and Cl- PEG silanes coated glass coverslips, while they remain confined on the patterned PLL-g-PEG coated glass coverslips.
CONCLUSIONS We have reported a simple, versatile and rapid method for protein and cell micropatterning on optimal antifouling surfaces. Using deep UV irradiation for both surface activation and surface patterning allows to completely eliminate any specialised equipment from the production of the micro-patterned surface, which can then be implemented in any biology lab at low cost. We believe that the reproducibility, easiness, versatility and low cost of this method, make it a method of choice for use in the lab, and could also lead to implementation of new micro-patterned culture surfaces, like multi-well microplates. The low production cost and long term storage resistance of such substrates might offer the possibility to make micropatterned cell culture substrates commercially available, opening the way to a widespread use in academic research and in industrial applications. ACKNOWLEDGEMENTS The work was supported by the translational research department of Institut Curie, and by the Agence National de la Recherche (ANR), France. REFERENCES [1] M. Théry, V. Racine, A. Pépin, M. Piel, Y. Chen, J-. B. Sibarita and M. Bornens, The extracellular matrix guides the orientation of the cell division, nature cell biology, pp. 947-953, (2005). [2] X. Jiang, D. A. Bruzewicz, A.P. Wong, M. Piel, and G. M. Whitesides, Directing cell migration with asymmetric micropatterns Directing cell migration with asymmetric micropatterns, PNAS, pp. 975-978, (2005). [3] Manuel Théry, V. Racine, M. Piel, A. Pépin, A. Dimitrov, Y. Chen, J-B. Sibarita and M. Bornens, Anisotropy of cell adhesive microenvironment governs cell internal organization and oentation of polarity,PNAS, pp. 19771–19776, (2006).
A. S. Blawas and W. M. Reichert, Protein patterning, Biomaterials, pp. 595609, (1998). [5] S. A Ruiz and C. S. Chen, Microcontact printing: A tool to pattern, Soft Matter, pp. 1–11, (2007). [6] J. Fink, M. Théry, A. Azioune, R. Dupont, F. Chatelain, M. Bornens and M. Piel, Comparative study and improvement of current cell micro-patterning techniques, Lab Chip, pp. 672–680, (2007). [7] L.G. Harrisa, S. Tosatti, M. Wieland, M. Textor, R.G. Richards, Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-functionalized poly(l-lysine)-grafted-poly(ethylene glycol) copolymers, Biomaterials, pp. 4135–4148, (2004). [8] J. Blümmel, N. Perschmann, D. Aydin, J. Drinjakovic, T. Surrey, M. LopezGarcia, H. Kessler and J. P. Spatz, Protein repellent properties of covalently attached PEG coatings on nanostructured SiO2-based interfaces, Biomaterials, pp. 4739–4747, (2007). [4]
