This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.
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.
All equipment and reagents have to be purchased sterile or needs to be heat or steam sterilized or sterilized with 70% ETOH.
The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Duisburg-Essen (Germany).
Part 1: Enucleation of murine eyes of different developmental stages
Enucleation of embryonic eyes
Enucleation of postnatal and adult eyes
Note: As at postnatal day 2, the orbital bones are still cartilaginous, it is important not to apply too much pressure while trying to remove the eyes.
By contrast, in the adult mice, the orbital bones are firm. Thus, in order to enucleate the eyes it is sufficient to apply pressure to the orbit without enlarging the eye slits in advance.
Part 2: Dissection of embryonic, postnatal and adult murine retinas
Dissection of retinas
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.
Part 3: Murine organotypic retinal wholemount culture
Part 4: Dissociation of cultured retinal wholemounts
Part 5: Washing of dissociated cell suspensions
Note: The addition of sodium acid allow storage of the cell suspension for several days at 4°C. However, if an immunocytochemical staining will follow, do not add sodium acid to the resuspension buffer, as this results in loss of staining quality.
Part 6: Cytospin of cell suspensions for quantitative apoptosis and proliferation analysis
Part 7: Representative Results
Figure 1: Steps in preparation of murine organotypic retinal wholemounts
A Head of mouse with both eyes. B Murine eye with lens side up, all layers still in place. C Murine eye from the backside with the optic nerve still attached. D Murine eye with sclera and pigment epithelium partially removed. E Murine retina with cornea, sclera and pigment epithelium completely removed, but lens and vitreous still in place. F Murine retinal wholemount cup with lens and vitreous removed. Please click here to see a larger version of figure 1.
Figure 2: Analysis of organotypic retinal wholemount cultures by cytospin and sections
For analysis of apoptosis, cytospins of dissociated cell suspensions are stained by DAPI and pycnotic nuclei can be distinguished by nuclear fragmentation or chromatin condensation (arrowheads in A). Alternatively, wholemount sections (C-E; murine retina postnatal day (P) 2) or retinal flatmount (F) can be subjected to a TUNEL assay and counterstained with DAPI (E). The effect of treatment on different retinal cell types can be visualized in cytospins by neuron specific antibodies like the ganglion cell marker Brn3a (arrows in B). GCL, ganglion cell layer; INL, prospective inner nuclear layer. Please click here to see a larger version of figure 2.
The advantage of murine organotypic retinal wholemount cultures2-5 over dissociation, monolayer, retinospheroid or re-aggregated 3D spheroid cultures1 lies in the preservation of neuronal interactions and connections, mimicking the in vivo situation. In comparison to former reports2, our video article provides a detailed demonstration of the peculiarities in enucleation of murine eyes and dissection of retinas of different developmental stages including removal of lens and vitreous body without damaging the retina. The removal of the lens and vitreous is essential for pharmacological manipulations as both hinder the access of substances to the retinal layers. In contrast to other reported murine explant culture systems, we do not use supportive materials, e.g. polycarbonate membranes2, for our organotypic culture, but culture the retinal wholemount cups free floating, additionally facilitating the accessibility for exogenously applied substances3-5.
Using a chemically defined, serum- and supplement-free culture medium without insulin allows only for short-time culture (24 – 48 hrs) but counterbalancing effects of insulin on apoptosis levels3 or growth factor-mimicking effects of FCS and supplements are avoided.
Most apoptosis and proliferations studies use MTT assays or FACS analysis for quantification of effects. We, however, provide a step by step demonstration of dissociation of cultured retinas for quantitative apoptosis and proliferation analysis by cytospin. As far as our experience goes, manual counting of nuclei in DAPI or BrDU stained cell spots – although tedious and time consuming – is the most accurate method for quantifying retinal apoptosis and proliferation, especially in murine retinas.
The authors would like to thank E. de la Rosa and A.I. Valenciano for initial help with the establishment of the organotypic cultures and U. Laub and U. Gerster for technical assistance.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Mice | Animal | Charles Rivers Laboratories | ||
Dissection microscope | Tool | ZEISS | ||
PBS | Reagent | Sigma | PBS should be cold (> 4°C) and sterile | |
Dulbecco`s modified eagle`s medium / nutrient mixture F-12 Ham | Reagent | Sigma | D 8900 | DMEM / F-12 |
Apo-transferin | Reagent | Sigma | T 1147 | |
Putrescin | Reagent | Sigma | P 5780 | |
Sodium selenite | Reagent | Sigma | S 9133 | |
Progesterone | Reagent | Sigma | P 6149 | |
Gentamicine | Reagent | Invitrogen | ||
L-Glutamine | Reagent | Invitrogen | 25030-024 | 200 mM (100X), liquid |
Bovine serum albumine (BSA) | Reagent | Roth | 8076.3 | 30 mg/ml |
Collagenase | Reagent | Sigma | C 0773 | 200 U/ml |
Trypsin | Reagent | Sigma | T4799 | From porcine pancreas; 1 mg/ml |
Hyaluronidase | Reagent | Sigma | H 3884 | 200 mg/ml |
DNase I | Reagent | Roche | 1 284 932 | 10 mg/ml |
EDTA | Reagent | Sigma | E 6511 | |
Silicone solution | Reagent | Serva | 35130 | |
Paraformaldehyde (PFA) | Reagent | Sigma | P6148 | 8% PFA in 0.1M phosphate buffer (pH 7.4). |
4′,6-diamidino-2-phenylindole dihydrochloride | Reagent | Sigma | D 0542 | DAPI |
Fluorescent Mounting Medium | Reagent | Dako | S3023 | |
BrDU | Reagent | Sigma | B 9285 | |
96-well plates | Tool | FALCON | 3072 | |
24-well plates | Tool | FALCON | 3047 | |
Pasteur pipettes | Tool | Brand | 747720 | |
Forceps DUMONT #5 | Tool | Fine Science Tools | 11252-30 | bevelled very fine shanks (0.05 mm x 0.02 mm tip) |
Forceps DUMONT #7 | Tool | Fine Science Tools | 11271-30 | curved shanks (0.07 mm x 0.10 mm tip) |
Spring scissors,straight, 8cm | Tool | Fine Science Tools | 15000-00 | fine, small straight blades |
Standard scissors, straight, sharp/blunt | Tool | Fine Science Tools | 14007-14 | Use for decapitation or cervical dislocation |
Eppendorf tubes | Tool | Eppendorf | 2ml; round bottom for better precipitation of pellet during centrifugation /cytospin | |
Cooling centrifuge | Tool | Eppendorf | ||
Rotation shaker | Tool | CAT | ||
Cytospin | Tool | Thermo Scientific |