Next we wanted to investigate how neurons grow within and across the perfusion-channel chamber and if the flow properties are maintained (as with the neuron-free chamber) when neurons are cultured for extended periods of time. Fourteen days after plating, we observed numerous dendrites extending through the perfusion channel and often extending into the corresponding microgrooves present across the perfusion channel (Figure S2). Dendrites also sometimes branched within the perfusion channel. We then perfused fluorescent dye within the local perfusion channel to determine if we could maintain a distinct perfusion stream, as previously observed in chambers lacking neurons (Fig 3B). We used chambers containing neurons over 14 days in culture and observed that the perfusate often diffused into the microgrooves during the perfusion (Figure S2). In most cases the diffusion occurred in microgrooves which had large accumulations of cellular material, suggesting that the diffusion of perfusate is due to blockage of flow within the microgrooves. These results indicate that the single perfusion stream design is not adequate to prevent diffusion of the perfusate into the microgrooves when neurons are cultured for extended periods of time.
