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

Kaempf, S.; Walter, Peter; Salz, A. K.; Thumann, Gabriele

doi:10.1016/j.jneumeth.2008.05.018

Cited by 54 publications

(53 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…18,[34][35][36] This protocol is well established and is used by most groups studying organ cultures of adult full-thickness porcine retina. 13,18,19,36 Thus, the biomechanical loss of tension without interference from lack of nutritional support can be studied in isolation. Under standard culture conditions, the isolated retinal full-thickness explanted sheet has lost much of its normal tension, but is supported to some extent by the culture membrane.…”

Section: Figure 6 Immunohistochemical Labeling Of Müller Cells Usingmentioning

confidence: 99%

Stretch to See: Lateral Tension Strongly Determines Cell Survival in Long-Term Cultures of Adult Porcine Retina

Taylor

Moran

Arnér

et al. 2013

Invest. Ophthalmol. Vis. Sci.

View full text Add to dashboard Cite

PURPOSE.To explore the effect of lateral tension as a survival factor for retinal explants in vitro. The central nervous system (CNS) resides in a highly mechanical milieu. However, the importance of biomechanical homeostasis for normal CNS function has not been extensively explored. Diseases in which normal mechanical forces are disrupted, such as retinal detachment of the eye, are highly debilitating and the mechanisms underlying disease progression are not fully understood.METHODS. Using a porcine animal model, we developed a novel technique of culturing adult retinal explants under stretch for up to 10 days in vitro (DIV). These were compared with standard (no stretch) and free-floating cultured explants. Cell survival was analyzed using immunohistochemistry, and retinal architecture using hematoxylin and eosin staining.RESULTS. Compared with unstretched specimens, which at 10 DIV degenerated into a gliotic cell mass, stretched retinas displayed a profound preservation of the laminar retinal architecture as well as significantly increased neuronal cell survival, with no signs of impending gliosis. CONCLUSIONS.The results confirm that biomechanical tension is a vital factor in the maintenance of retinal tissue integrity, and suggest that mechanical cues are important components of pathologic responses within the CNS. (Invest Ophthalmol Vis

show abstract

Section: Figure 6 Immunohistochemical Labeling Of Müller Cells Usingmentioning

confidence: 99%

Stretch to See: Lateral Tension Strongly Determines Cell Survival in Long-Term Cultures of Adult Porcine Retina

Taylor

Moran

Arnér

et al. 2013

Invest. Ophthalmol. Vis. Sci.

View full text Add to dashboard Cite

show abstract

“…In addition, each explant required its own perfusion system making in vitro culture of multiple tissue samples cumbersome. Kaempf et al successfully created another organotypic culture model of adult mammalian neurosensory retina in co-culture with RPE but only showed results for a 3-day culture; the long-term survival of the tissue was not tested 8 .…”

Section: Discussionmentioning

confidence: 99%

Organotypic Culture of Full-thickness Adult Porcine Retina

Wang

Kolomeyer

Zarbin

et al. 2011

JoVE

View full text Add to dashboard Cite

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina. Protocol Making a custom-made stand for cell culture insertsThe base of a 5 ml pipette tip (ISC BioExpress, #P-3250-19) has an inner diameter of 13 mm, which perfectly matches the bottom size (12.6 mm, outer diameter) of a 10 mm (inner diameter) NUNC cell culture insert (8 μm, Polycarbonate membrane, Thermal, #137443). To make the stand, the pipette was cut off near the base to produce a 6 mm long hollow cylinder. Then, pieces were removed from the side of the cylinder using a flame-heated blade to create 3 legs (4 mm in height) under a ring (2 mm in height), which raised the height of the inserts by approximately 5 mm. Preparation of sterile filter paper for attachment and culture of porcine retinaWhatman 4M Filter paper (Cat No. 1004) was cut into a circular shape (6.3 mm in diameter) with a small, triangular handle that was used for moving the filter paper into a glass tube for sterilization or once the retina was attached (as shown in the video). Preparation of retinal explantsEyes from 5-to 9-month old, 150-230 lbs, American Yorkshire pigs were obtained from a local abattoir within three hours of enucleation (transported on ice). Upon arrival, eyes were cleaned of extraneous tissue, dipped in betadine solution (10% Povidone-iodine, The Purdue Frederique Company, Stamford, CT) and washed twice in Dulbecco's Modified Eagle Medium (DMEM with 1g/L glucose, L-glutamine, and sodium pyruvate, Cellgro-mediatech Inc., Manasses, VA) supplemented with 2.5 μg/ml Amphotericin B (Gibco-Invitrogen, Carlsbad, CA). The anterior segment was removed, exposing the neural retina. Six-mm trephine blades (Storz Ophthalmic-Bausch and Lomb, Manchester, MO) were used to cut equatorial, full-thickness posterior segment explants. The retina was subsequently peeled off by gently applying a piece of dry sterile filter paper onto the ganglion cell layer, lifting o...

show abstract

“…Isolated adult retinal sheets cultured under standard conditions often display gliosis and neuronal degeneration over time. The discrepancy of cell survival in vitro, depending on the stage of maturity of the retinal tissue, is well established in the literature but not yet fully understood [Fernandez-Bueno et al, 2008;Kaempf et al, 2008;Kobuch et al, 2008;Taylor et al, 2013a]. Nevertheless, retina explant cultures have been widely used to study neurodegeneration, neuroprotection, and various applications [Hatakeyama and Kageyama, 2002;Manabe et al, 2002;Zhang et al, 2002;Donovan and Dyer, 2006;Leaver et al, 2006;Koizumi et al, 2007;Xin et al, 2007;Bermel et al, 2009;Feigenspan et al, 2010].…”

Section: Ex Vivo Retina Explant Culturesmentioning

confidence: 99%

“…Posterior segment explant: retina, retinal pigment epithelium, choroid, sclera [Kaempf et al, 2008;Denk et al, 2015] Vitreoretinal explant [Peynshaert et al, 2017] Inner retina or GCL facing up (most common) GCL facing down [Wang et al, 2011;Taylor et al, 2014] Note: Retina explants are commonly maintained at the air-liquid interface, except for the study by Wang et al [2011], which cultured the explants completely submerged in cell culture media…”

Section: Retinal Microarchitecture and Morphometric Analysismentioning

confidence: 99%

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems

Rettinger

Wang²

2018

Cells Tissues Organs

View full text Add to dashboard Cite

Retinal degenerative diseases such as macular degeneration, glaucoma, and diabetic retinopathy constitute the leading cause of blindness in the industrialized world. There is a continuous demand in investigative ophthalmic research for the development of new treatment modalities for retinal therapy. Unfortunately, efforts to identify novel neuroprotective and neuroregenerative agents have often been hindered by an experimental model gap that exists between high-throughput methods via dissociated cells and preclinical animal models. Even though dissociated cell culture is rapid and high-throughput, it is limited in its ability to reproduce the in vivo conditions. In contrast, preclinical animal models may offer greater fidelity, albeit they lack efficiency and experimental control. Retina explant cultures provide an ideal bridge to close this gap and have been used to study an array of biological processes such as retinal development and neurodegeneration. However, it is often difficult to interpret findings from these studies due to the wide variety of experimental species and culture methods used. This review provides a comprehensive overview of current ex vivo neuroretina culture methods and assessments, with a focus on their suitability, advantages, and disadvantages, along with novel insights and perspectives on the organotypic culture model as a high-throughput platform for screening promising molecules for retinal regeneration.

show abstract

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

Cited by 54 publications

References 73 publications

Stretch to See: Lateral Tension Strongly Determines Cell Survival in Long-Term Cultures of Adult Porcine Retina

Stretch to See: Lateral Tension Strongly Determines Cell Survival in Long-Term Cultures of Adult Porcine Retina

Organotypic Culture of Full-thickness Adult Porcine Retina

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems

Contact Info

Product

Resources

About