Low density culture of mammalian primary neurons in compartmentalized microfluidic devices

This paper demonstrates the fabrication of a compartmentalized microfluidic device with docking sites to position a single neuron or a cluster of 5-6 neurons along with varying length of microgrooves and the optimization process for culturing primary mammalian neurons at low densities. The principle of centrifugation was employed to situate cells in desired locations followed by the application of a fluid flow to remove the extra or unwanted cells lying in the vicinity of the located neurons. The neuronal cell density was optimized by seeding 10³ cells and 10⁴ cells/microfluidic device. The speed of centrifugation was optimized as 1500 rpm for 1 min and a cell density of greater than or equal to 10⁴ cells/microfluidic device was found to be suitable for loading maximum number of docking sites. The outcomes of the simulated experiments was found to be in compliance with the experimemtal verifications. Furthermore, the cells cultured within the microfluidic device were assessed for immunocytochemical staining and the axonal growth was quantified with the help of an Axofluidic software. Although, several in vitro microfluidic platforms have been developed that facilitate the investigations where communication between neurons or between neurons and other cell types is concerned, none of the partitioned devices so far has reported the presence of docking sites along with an array of grooves of varying lengths. These physically connected but fluidically isolated compartmentalized microfluidic devices may serve us in analysing the activity of a low density of neurons and the influence of axonal length in setting up a communication with other cell type.This platform is useful to gain insights into the processes of synapse formation, axonal guidance, cell-cell interaction, to name a few.