In vivo-like Organotypic Murine Retinal Wholemount Culture

Summary

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.

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 preserved^2-5.

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.

Protocol

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)…

Discussion

The advantage of murine organotypic retinal wholemount cultures^2-5 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 vitreo…

Materials

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