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Matthias Strake, Florian Rehfeldt, Christina Stanischa, Peer Lauermann, Hans Hoerauf, Christian van Oterendorp; The effect of laminar flow on cultured retinal ganglion cell survival and neurite outgrowth.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5295.
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© ARVO (1962-2015); The Authors (2016-present)
Applying fluidic flow to cultured neurons is an established method to guide and modify neurite outgrowth. We tested this approach on primary cultures of retinal ganglion cells (RGC) and analysed the influence of low velocity laminar flow on neurite morphology and cell survival.
Primary cultures of retinal cells were set up using p2-7 Wistar rat retinae. Cells were seeded into a micro slide connected to a fluidic pump (Ibidi, Germany). After 6 hours of no-flow-incubation a constant laminar flow inside the slide was started. The flow velocities applied were 4.1*10^-4±9.6*10^-5 mm/s, 0.12±3.8*10^-2 mm/s and 1.8±4.5*10^-2 mm/s (mean ± SD) between experiments. After 2 days, cells were fixated and immunohistochemically stained for Beta-III-tubulin to identify RGC. The RGC number and total neurite length per cell was analysed with ImageJ software. For neurite length analysis only cells with at least one visible neurite were selected.
Compared to no-flow control cultures, the application of 0.12 mm/s flow velocity highly significantly reduced both, RGC density (0.9710±0.4523 vs 0.199±0.3413 cells/mm2; p=0.0005; mean±SD) and neurite outgrowth (total neurite length per cell: 0.12 mm/s flow: 22.45±21.70, n=24 vs. no flow: 73.27±54.65, n=44 (p<0.0001)). The lowest flow rate (4*10^-4 mm/s) led to significantly higher RGC density (0.5867±0.4502 cells/mm2, n=31, p=0.0412) and neurite outgrowth (47.34±30.45 µm, n=66, p=0.0003). Between the lowest flow and no-flow the differences were not statistically significant (p=0.63 and 0.24, respectively). Increasing the flow rate from 0.12 mm/s to 1.8 mm/s did not further impair cell density and neurite outgrowth. However, the both parameters were already close to zero at 0.12 mm/s.
Conclusions: Unlike other primary neuronal cultures, microfluidic flow is not suitable to guide neurite outgrowth in RGC. Even low flow velocities (<1 mm/s) significantly impaired cell survival and neurite length.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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