This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation
Natalie Bull
Keith Martin University of Cambridge (UK)
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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.
Keywords
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
This is a list of supplementary files associated with this preprint. Click to download.
- ExplantprotocolIntro.pdf
Introduction Introduction
- ExplantprotocolReagents.pdf
Reagents Reagents
- ExplantprotocolEquipment.pdf
Equipment Equipment
- ExplantProtocolProcedure.pdf
Procedure Procedure
- ExplantProtocolTroubleshooting.pdf
Troubleshooting Troubleshooting
- ExplantProtocolTiming.pdf
Timing Timing
- ExplantProtocolAnticipatedResults.pdf
Anticipated Results Anticipated Results
- ExplantProtocolReferences.pdf
References References
- ExplantProtocolAcknowledgements.pdf
Acknowledgements Acknowledgements
- MartinLabretinalexplantprotocol.pdf
Retinal Explant Protocol PDF Complete PDF of Martin Lab Retinal Explant Protocol
Version History
CURRENT STATUS: Posted
Version 1
Posted 04 Mar, 2011
Metrics
Comments: 0
HTML Views: ...
Associated Publications
Use of an adult retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies
by N. D. Bull, T. V. Johnson, G. Welsapar, +3
Investigative Ophthalmology & Visual Science (02 March, 2011)
Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an In Vitro Intraocular Stem Cell Transplantation Model
by T. V. Johnson and K. R. Martin
Investigative Ophthalmology & Visual Science (02 March, 2011)
Identification of Barriers to Retinal Engraftment of Transplanted Stem Cells
by T. V. Johnson, N. D. Bull, and K. R. Martin
Investigative Ophthalmology & Visual Science (02 March, 2011)
Method Article
Organotypic explant culture of adult rat retina for in vitro investigations of neurodegeneration, neuroprotection and cell transplantation
Natalie Bull
Keith Martin University of Cambridge (UK)
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 04 Mar, 2011
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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.
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
Associated Publications
Use of an adult retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies
by N. D. Bull, T. V. Johnson, G. Welsapar, +3
Investigative Ophthalmology & Visual Science (02 March, 2011)
Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an In Vitro Intraocular Stem Cell Transplantation Model
by T. V. Johnson and K. R. Martin
Investigative Ophthalmology & Visual Science (02 March, 2011)
Identification of Barriers to Retinal Engraftment of Transplanted Stem Cells
by T. V. Johnson, N. D. Bull, and K. R. Martin
Investigative Ophthalmology & Visual Science (02 March, 2011)