Adult zebrafish retinal cell culture in a microfluidic setup at DIV 3. (A) Adult zebrafish retinal neurons cultured in SDN450 MFDs, display limited outgrowth and network formation. Live cell images taken at DIV 3 demonstrate that Tg(Tru.gap43:GFP)mil1 (gap43) retinal neurons, loaded in the wells and channel, do not attach properly into the channel, which causes a flow of neurons from the channel toward the wells after seeding (DIV 0) or addition of medium (DIV 0–3). As a result of this low density in the SDC channel, neurons display strongly reduced neurite outgrowth and network formation compared to neurons growing in the SDC wells, and only a few neurites grow into the microgrooves at DIV 3. (B) In contrast, at DIV 3, gap43 retinal neurons seeded in the SDC of an open compartment SOC450 MFD form an extensive neuronal network throughout the entire SDC, including the areas bordering the microgrooves. Consequently, spontaneous RGC outgrowth into the microgrooves is achieved in this setup, with numerous axons growing far into the AC at DIV 3. Scale bars: 500 μm (overview pictures), 100 μm (detailed pictures). AC, axonal compartment; CNB, complete neurobasal medium; DIV, days in vitro; MFD, microfluidic device; RGC, retinal ganglion cell; SDC, somatodendritic compartment.
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