Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation

doi:10.1038/protex.2011.215

Method Article

Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation

https://doi.org/10.1038/protex.2011.215

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This protocol details a method for isolating retinal tissue from adult rats as an organotypic culture to study neurobiological processes in mature tissue. It combines the efficiency and control common to in vitro techniques with close imitation of the in vivo environment. Eyes from adult rats are enucleated and the neural retina is isolated. Tissue is cut into quarters, yielding eight retinal explants per animal, and cultured at a fluid/air interface on organotypic culture membranes. Explantation can be accomplished in thirty minutes per animal. Tissue is nourished by a serum-free medium and can be viably maintained for at least two weeks ex vivo. Protocols are provided that describe histological processing, including techniques for whole-mount and cross-sectional immunohistochemical labelling. In addition, methods for simulating intraocular cell transplantation and pharmacological screening for neuroprotective therapies are also provided.

Neuroscience

Biological techniques

neuroprotection

ganglion cell

organotypic culture

pharmacological screening

See PDF file "Explant Protocol Introduction":http://www.nature.com/protocolexchange/system/uploads/1738/original/Explant_protocol_Intro.pdf?1299089296

For link to complete protocol see "Martin Lab Retinal Explant Protocol":http://www.nature.com/protocolexchange/system/uploads/1747/original/Martin_Lab_retinal_explant_protocol.pdf?1299090215

See PDF file "Explant Protocol Reagents":http://www.nature.com/protocolexchange/system/uploads/1739/original/Explant_protocol_Reagents.pdf?1299089329

See PDF file "Explant Protocol Equipment":http://www.nature.com/protocolexchange/system/uploads/1740/original/Explant_protocol_Equipment.pdf?1299089355

See PDF file "Explant Protocol Procedure":http://www.nature.com/protocolexchange/system/uploads/1741/original/Explant_Protocol_Procedure.pdf?1299089460

This document includes full details for performing the following steps:

Prepare an organotypic adult retinal explant culture and maintain this culture. TIMING: 45-60 minutes per rat (i.e. pair of eyes) for tissue dissection; 20-30 minutes for bi-daily medium exchanges
Co-culture retinal explants with dissociated cell types (e.g. stem cells). TIMING: cell transplantation 15-30 minutes (add approximately 10 minutes more for each additional plate).
Perform the pharmacological screen of neuroprotective drugs using retinal explants. TIMING: treating explants approximately 10 minutes per plate
Preserve the retinal explants for histological analysis. TIMING: approximately 24 hour
Perform cryostat sectioning of the retinal explants and label the sections by fluorescent immunohistochemistry. TIMING: cryoprotection and embedding ~24 hours; cryostat each explant block approximately 40-60 minutes; immunohistochemistry requires 2 days.
Perform fluorescent immunohistochemical labelling of the whole-mount explants. TIMING: approximately 3 days

See PDF file "Explant Protocol Timing":http://www.nature.com/protocolexchange/system/uploads/1743/original/Explant_Protocol_Timing.pdf?1299089510

See PDF file "Explant Protocol Troubleshooting":http://www.nature.com/protocolexchange/system/uploads/1742/original/Explant_Protocol_Troubleshooting.pdf?1299089491

See PDF file "Explant Protocol Anticipated Results":http://www.nature.com/protocolexchange/system/uploads/1744/original/Explant_Protocol_Anticipated_Results.pdf?1299089550

See PDf file "Explant Protocol References":http://www.nature.com/protocolexchange/system/uploads/1745/original/Explant_Protocol_References.pdf?1299089572

See PDF file "Explant Protocol Acknowledgements":http://www.nature.com/protocolexchange/system/uploads/1746/original/Explant_Protocol_Acknowledgements.pdf?1299089597

The authors declare no competing financial interests.

supplement0.pdf
Retinal Explant Protocol PDF Complete PDF of Martin Lab Retinal Explant Protocol
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Reagents Reagents
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Troubleshooting Troubleshooting
supplement0.pdf
Equipment Equipment
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Introduction Introduction
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Timing Timing
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Anticipated Results Anticipated Results
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References References
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Acknowledgements Acknowledgements
supplement0.pdf
Procedure Procedure

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Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation

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Version 1

Abstract

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

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