Roller Culture of Free-Floating Retinal Slices: A New System of Organotypic Cultures of Adult Rat Retina

Abstract

No experimental system exists to date for the in vitro study of retinal ganglion cell populations in a three-dimensional organotypic tissue environment. Here, we describe such a novel method for roller cultivation of adult retinas. Retinas of adult (1–3 months old) rats were cut into rectangular slices of approximately 1 mm². Free-floating slices were cultured on a horizontal rotating roller drum (50–60 rpm) in a dry incubator at 36.5°C. During the first days of cultivation, primary flat retinal slices changed their configuration and transformed into ball-shaped tissue spheres (retinal bodies). Histological and immunocytochemical studies showed that the outer wall of the retinal bodies was formed by cell and fibre layers typical of mature retina with photoreceptors located on the outside. Initially, retinal bodies contained an inner cavity which later was completely obliterated and filled with glial cells, sprouting nerve fibres, and vascular structures. This culture system was further developed into a robust model of glutamate-induced neurotoxicity. Using a novel culture method of adult rat retina, preservation of the three-dimensional organotypic retinal cytoarchitecture was achieved, including survival of neurons in the ganglion cell layer and sprouting of nerve fibres of the axotomized retinal ganglion cells. This novel culture model promises to facilitate studies of retinal physiology and pathology.

New method of in vitro culturing of pigment retinal epithelium in the structure of the posterior eye sector of adult rat

“…Indeed, the number of pyknotic cells in PRE during roller culturing in the structure of PEW without medium replacement did not exceed 10-20% until day 10, whereas during stationary culturing with daily medium replacement this parameter was by 3-fold higher (57%) as soon as on day 3 (maximum life time of PRE in the structure of PEW under these conditions). Studies on the retina isolated from rats [25,28] and mice [19,29] showed that rotation provides not only constant access of fresh medium to the preparation, but also leads to the formation of closed spheres from tissue or its sections. This, in turn, preserves layer-by-layer organization of the retina and cell-cell interactions.…”

“…Organotypic culturing is used for evaluation of the neural retina from eyes of adult mammals and embryos [19,23,25,28,29]. High viability and maintenance of the basic layer-by-layer structure of the neural retina in neonatal and early postnatal rodents and the possibility of its further development in vitro were reported.…”

The ‘Free-Floating’ Retina Epoch: New Models for New Scopes

“…On the other hand, cultures of complex tissues which permit cellular interactions in a fashion mimicking the in vivo conditions are certainly more valuable than dissociated cell cultures or cell lines.The paper published in the present issue by Rzeczinski and Victorov et al [2] shows that the authors have succeeded in establishing the roller culture of free-floating adult retina, a procedure that may be suitable for other vertebrate retinas too. When viewed from the clinicalpathological standpoint, the model appears promising for examining diseases such as glaucoma, hereditary retinal dystrophies or proliferative diseases like diabetic retinopathy.…”

Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium

“…The controlled environment in cell and tissue culture provides conditions for the analysis of individual biological mechanisms such as the action of neurotrophic factors or the function of synapses (Goureau et al, 2004;Doonan et al, 2005;Lagreze et al, 2005). However, the main difficulty in studies of adult neural tissues is the limited survival in culture (Germer et al, 1998;Winkler et al, 2000;Garcia et al, 2002;Ito et al, 2004;Rzeczinski et al, 2006).…”

“…Tansley (1933) advanced the technique by extending the method to mammalian tissue. Retinal explant cultures in the first half of the 20th century were primarily grown in plasma clots or in a collagen matrix using a roller tube method, also known as the flying coverslip method and variations on this method are still used today (Hild and Callas, 1967;LaVail and Hild, 1971;Feigenspan et al, 1993;Rothermel et al, 2005;Rzeczinski et al, 2006). In the 1950s Trowell (1954) developed the membrane culture in which the tissue is placed on a porous membrane on top of a wire grid and maintained at the air-medium interface.…”