Incubation of adult marmoset monkey retina with transient gene transfection.

<p>(A) Organotypic culture of adult marmoset retina after 42-hour incubation. Left, a photograph of the interphase chamber of our organotypic culture system. Right, a schematic diagram of the interphase chamber. Deep dishes were used with custom-made stands to support the tissue culture insert with a flat-mounted retina. The retina was in contact with the medium over the filter on the photoreceptor side, and the ganglion cell side faced the atmosphere. Dishes were agitated by a rotary shaker in the CO₂ incubator during the incubation. (B) Patch-clamp recordings of a cultured retinal ganglion cell after 72-hour incubation. Left: Voltage-clamp recordings. Holding voltage = −71 mV. Voltages were clamped from −91 mV to +19 mV, in 10-mV steps, for 100 msec (bar). Right: Current-clamp experiment. Injected currents were from −20 pA to 50 pA, in 10-pA steps, for 100 msec (bar). Resting membrane potential was −59 mV. (C) A piece of marmoset retina after 3-day culture was stained with YO-PRO-1. YO-PRO-1-positive cells were clustered on the retina piece. The clusters formed an area with bright green fluorescence so that we defined a clustered area as a YO-PRO-1-positive area (black areas in left panel). Scale bars, 1 mm. (D) Quantification of YO-PRO-1-positive areas on cultured marmoset retinas. In the cultured retinas, 18.6±9.8% (mean ± standard deviation, n = 4 retina pieces) of the whole retinal area was YO-PRO-1-positive, while 22.0±6.0% (n = 4) of the retinal area was also YO-PRO-1-positive even in the acutely isolated retina (no significant difference). (E) A piece of <i>GFP-F</i>-transfected retina after 42-hour incubation. In this piece of retina, there were more than 452 cells expressing GFP-F. Scale bar, 1 mm. (F) Another piece of <i>GFP-F</i>-transfected retina after 42-hour incubation. Several retinal ganglion cells with different morphologies expressed GFP-F. Scale bar, 100 μm.