Micropatterning neural cell cultures in 3D with a multi-layered scaffold Anja Kunze a, * , Michele Giugliano b, c , Ana Valero a , Philippe Renaud a a Microsystems Laboratory (LMIS4), Institute of Microengineering Lausanne, Ecole Polytechnique Fédérale de Lausanne, Station 17, CH-1015 Lausanne, Switzerland b Neural Microcircuitry Laboratory, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, Station 19, CH-1015 Lausanne, Switzerland c Department of Biomedical Sciences, University of Antwerp, B-2610 Wilrijk, Belgium article info Article history: Received 1 November 2010 Accepted 19 November 2010 Available online 14 December 2010 Keywords: Micropatterning Agarose-alginate hydrogel Multi-layered scaffold Primary neural cell culture Neurite outgrowth abstract Cortical neurons, in their native state, are organized in six different cell layers; and the thickness of the cell layer ranges from 0.12 mm to 0.4 mm. The structure of cell layers plays an important role in neurodegenerative diseases or corticogenesis. We developed a 3D microfluidic device for creating physiologically realistic, micrometer scaled neural cell layers. Using this device, we demonstrated that (1) agaroseealginate mixture can be gelled thermally, thus an excellent candidate for forming multi-layered scaffolds for micropatterning embedded cells; (2) primary cortical neurons were cultured successfully for up to three weeks in the micropatterned multi-layered scaffold; (3) B27 concentration gradient enhanced neurite outgrowth. In addition, this device is compatible with optical microscopy, the dynamic process of neural growth can be imaged, and density and number of neurites can be quantified. This device can potentially be used for drug development, as well as research in basic neural biology. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction In the last decades, neuroscientists have debated on the funda- mental structure, formation and function of the cerebral cortex of mammalian brains [1e3]. The final developed structure of the cerebral cortex appears in a strongly layered formation; the layers are defined by their form and dimension, cell behavior and fate of cells. Generally, the cerebral cortex consists of six cell layers. The cell layers are defined from the outer part of the cortex to the inner part: the first layer, called molecular layer, is a cell-free layer containing only axons and dendrites; the second contains small spherical granular cells; the third is the first to contain pyramidal neurons; the fourth layer consists of again granular cells and separates the external from the internal pyramidal cells, which are later being found in the fifth layer; and finally the last, the sixth cell layer, is a heterogeneous layer of neurons, carrying mainly axons to and from the cortex [4]. If wrong laminar cell formation appears during the development of the cortex, the so-called corticogenesis, mental disorder diseases can occur. The laminar-layered structure of the cortex might also play an important role in neurodegenerative diseases. Today, drug screenings, toxic tests and developmental studies in vitro are still based on homogeneously seeded, dissociated neural cell cultures [5e7]. Dissociated neural cell cultures, however, provide an easy to handle cell model, but are reduced to a 2D environment by the Petri-dish confinement, constraining neural cells to behave differently, compared to their in vivo environment [8]. One way to overcome structuring restrictions is to pattern cells on 2D surfaces with cell patterning methods [9]. Here, we focus only on micro cell patterning methods; ranging from selective cell attach- ment on micro- and nanostructures over selective protein absorption by micro contact printing, photolithography or microfluidic based surface gradient patterning [10]. Neural cells have already been successfully structured using micro contact printing and surface gradient generation of laminin, or by anchoring them on micro- structures [11e 13]. 2D Cell patterning methods provide a very controllable structuring process of neural cells, but they are restricted to 2D surfaces. Furthermore, it remains open, what kind, shape, or formation of the 2D pattern represents neural functionality close to the in vivo cell environment. Hence, there is a need to define a multi- layered scaffold close to the natural micro scale of neural cell layers for micropatterning and culturing primary cortical neurons in 3D. Most commonly used materials for a 3D cell culture scaffolds are hydrogels [14]. Hydrogels can be divided by their origin into natural and synthetic hydrogels [15]. In our paper we focus only on natural hydrogels, because they are cheaper and easier to produce. In the past, neural cells have already been cultured in agarose, alginate, collagen and matrigel [16e20]. For this paper we chose to culture primary cortical neurons in an agarose based scaffold, because of the well-known mechanical properties and the ability to form a gel within seconds. Re-engineering a 3D layered structure has already been tried with other cell types such as fibroblast or endothelial cell lines, but * Corresponding author. Tel.: þ41 21 693 6839; fax: þ41 21 693 5950. E-mail address: anja.kunze@epfl.ch (Anja Kunze). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.11.047 Biomaterials 32 (2011) 2088e2098