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

• Protocol
• Discussion

Authors: Natalie Bull,Thomas Johnson & Keith Martin

Abstract

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.

Introduction

See PDF file Explant Protocol Introduction

For link to complete protocol see Martin Lab Retinal Explant Protocol

Reagents

See PDF file Explant Protocol Reagents

Equipment

See PDF file Explant Protocol Equipment

Procedure

See PDF file Explant Protocol Procedure

This document includes full details for performing the following steps:

1. 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
2. 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).
3. Perform the pharmacological screen of neuroprotective drugs using retinal explants.
   • TIMING: treating explants approximately 10 minutes per plate
4. Preserve the retinal explants for histological analysis.
   • TIMING: approximately 24 hour
5. 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.
6. Perform fluorescent immunohistochemical labelling of the whole-mount explants.
   • TIMING: approximately 3 days

Troubleshooting

See PDF file Explant Protocol Troubleshooting

Anticipated Results

See PDF file Explant Protocol Anticipated Results

References

See PDf file Explant Protocol References

Acknowledgements

See PDF file Explant Protocol Acknowledgements

Figures

Retinal Explant Protocol PDF: Complete PDF of Martin Lab Retinal Explant Protocol

Download Retinal Explant Protocol PDF

Associated Publications

1. Use of an adult retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies. N. D. Bull, T. V. Johnson, G. Welsapar, N. W. DeKorver, S. I. Tomarev, and K. R. Martin. Investigative Ophthalmology & Visual Science 23/02/2011 doi:10.1167/iovs.10-6873
2. Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an In Vitro Intraocular Stem Cell Transplantation Model. T. V. Johnson and K. R. Martin. Investigative Ophthalmology & Visual Science 49 (8) 3503 - 3512 03/03/2008 doi:10.1167/iovs.07-1601
3. Identification of Barriers to Retinal Engraftment of Transplanted Stem Cells. T. V. Johnson, N. D. Bull, and K. R. Martin. Investigative Ophthalmology & Visual Science 51 (2) 960 - 970 01/02/2010 doi:10.1167/iovs.09-3884

Author information

Natalie Bull, Keith Martin University of Cambridge (UK)

Thomas Johnson, Johns Hopkins University

Keith Martin, University of Cambridge (UK)

Correspondence to: Natalie Bull ([email protected])

Source: Protocol Exchange (2011) doi:10.1038/protex.2011.215. Originally published online 4 March 2011.