Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery

Abstract

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

Figure 1

This illustration depicts the surgical preparation of freshly enucleated mouse eyes that were used for viral transduction.

Figure 2

Three-dimensional reconstruction of confocal microscopy of a retinal section counterstained with the nuclear marker DAPI, displaying the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL).

Figure 3

Photomicrograph of a hematoxylin and eosin stained retinal explant at day 2 ex vivo at 10× (A) and 40× (B) magnification, displaying the retinal layers and adjacent choroid and sclera.

Figure 4

A — Percentage of nuclei within the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL), compared to clinically normal eyes in vivo (equalling 100%). With the exception of day 0, for each day ex vivo the values represent means of 16 counts: from 4 slides prepared from each of 4 mice. For day 0, 4 slides were prepared from 2 mice. Bars represent standard deviation from the mean. B — Photomicrographs of retinal explant sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI) representative of days 1, 4, and 7 ex vivo.

Figure 5

Percentage of apoptotic cells compared to the absolute nuclei count per area in the different retinal cell layers [ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL)] over the culture period days 0 to 7 ex vivo. Values represent mean counts for each layer from eye cups from 2 different mice. Bars represent standard deviation from the mean.

Figure 6

The 5 × 10⁸ or 5 × 10⁹ transduction units of vector expressing green fluorescent protein (GFP). Percentage of cells expressing the transduced gene compared to absolute numbers of cells per layer. Transduction efficiency was consistently higher in retinal cells of mice of the younger age group than the older age group. Note that the transduction efficiency was significantly higher in the outer nuclear layer (ONL) than the inner nuclear layer (INL) or ganglion cell layer (GCL) in both age groups.