Advanced Methods for Culturing Neuronal Cells with Microstructures

Abstract

During the development of the neural tissue of the nervous system, supporting neuroglial cells and the extracellular matrix (ECM) guide the migration of the immature functional neurons, provide them with a scaffold to grow on and aid in the formation of synapses. ECM provides the neurons with various guidance cues that guide the migration of the neurons and the extension of the neurites. The micrometre and even nanometre guidance cues can also be incorporated into neuronal cultures in vitro to study the effect of the guidance cues and to develop small neuronal networks with a desired architecture. Three-dimensional neurocage structures were fabricated from Ormocomp®, a polymer-ceramic hybrid material, by two-photon polymerisation for this study. In this study these structures were tested with neuronal cells differentiated from human embryonic stem cells (hESC). The neurocages were attached to microscope glass slide samples, each sample containing approximately ten neurocages. The neurocages were first coated with laminin, an ECM protein, to enable the adhesion of the cells to the glass surface. The cell suspension was then applied and the cells were cultured to observe their growth and to study the guidance effects of the neurocages. During the study eight individual experiments were carried out to optimise both the application methods for the laminin solution and the cell suspension and the growth of the cells. This study utilised both simple manual methods and two different micromanipulator set-ups in the application of the laminin solution and the cell suspension. It was concluded that the best method to apply the laminin solution was the micromanipulator set-up SU2, which utilised automated micromanipulator placement and pressure regulation. The cell suspension, on the other hand, could be easily applied onto the samples by placing droplets of the solution onto the medium covering each sample. As the cell population in the culture was small, conditioned medium taken from another neuronal cell culture was tested to increase the viability of the cell cultures of this study. The conditioned medium had a clear positive effect and the use of conditioned medium in future studies is therefore recommended. During the study it was found that the cells initially inside the neurocages did not attach to the glass bottom even when the insides of the neurocages were accurately coated with the laminin solution. In contrast, the cells outside the neurocages generally attached to the glass bottom well and had a tendency to migrate towards the neurocages and over the structure walls into them. Therefore, it was concluded that the material Ormocomp® was not harmful to neuronal cells and it even seemed to attract them. Furthermore, the cells that had migrated into the neurocages readily stayed inside them and extended neurites along the edges of the neurocages. This indicated that the neurite guidance properties of the structures were very promising. /Kir11