Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

Abstract

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2-9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day. The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

Cytotoxicity of β-Cyclodextrins in Retinal Explants for Intravitreal Drug Formulations

“…To study the cytotoxicity of CDs on the retinal tissue, retinas from mice were isolated for culturing for an extended period of time. The detailed protocol is described elsewhere [ 55 ], but will be summarized here. Immediately after animal sacrifice, the eyes were enucleated and incubated for 5 min at room temperature (RT) in R16 serum-free culture medium (Gibco, Carlsbad, CA, USA).…”

“…The retina with the retinal pigment epithelium (RPE) attached was isolated and cultured on a Transwell membrane (polycarbonate, 0.4 µm pore size, COSTAR, NY) with the RPE side facing down in a 6-well plate. Then, 1 mL complete medium (CM, R16 medium with supplements; detailed under [ 55 ]) was added to each well. The explants were allowed to recover from the explantation procedure in a sterile incubator (37 °C, 5% CO 2 ) for 48 h. The CM was exchanged every second day by removing 0.7 mL of the CM in the plate and adding 0.9 mL fresh CM to account for evaporation and conserve neuroprotective agents produced by the retinal culture.…”

Nature-inspired wax-coated jute bags for reducing post-harvest storage losses

“…Principles underlying water repellency in various animals and plants have been harnessed for developing technologies for separation and purification 64 , 71 , desalination 72 – 74 , cavitation mitigation 75 , coating-free entrapment of air underwater 50 , 76 , 77 , and producing super-water-repellent cotton 56 , among others 65 . However, their application in agricultural engineering and technology has remained limited 78 , 79 .…”

Efficient Hydrophilic Small Molecule Delivery To Retinal Cell Lines Using Solid Lipid Nanoparticle Containing Gel Core

“…While the relatively simple cell culture environment yielded interesting data, a full drug/DDS efficacy testing will likely require more complex test systems. More advanced in vitro tests using organotypic retinal explant cultures, in which the normal histotypic context of the retina is preserved [38], or in vivo injections will further characterize the suitability of the new SLPs for the delivery to the retina. Nevertheless, based on our formulation development and initial validation studies, this DDS appears to be suitable for intraocular administration of small hydrophilic compound for the treatment of retinal diseases.…”