Abstract
Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.
Highlights
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment
Important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures[1] are used
Organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved[2,3,4,5]. In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors
Summary
5. The embryos are separated, transferred to a Petri dish and uterus wall and embryonic membranes are removed carefully by the use of forceps. 2. To remove the outer eye layers, in postnatal stages and adult eyes the optic nerve has to be cut off by the help of spring scissors or pinched off by forceps as close to the basis as possible. Enter the subretinal space between retina and pigment epithelium with the tips of two very fine forceps from the site where the optic nerve penetrated the eye layers. 5. Peel off the layers up to the level of the cornea, turn the eye cup to the lens side and remove the cornea together with pigment epithelium, choroid membrane and sclera, while holding up the remaining retinal cup by the other forceps. Note: Between dissection of individual retinas, keep the 96-well collection plate containing the culture medium in the incubator as the pH of the culture medium is triggered by CO2 via the carbonate system
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