Optimized culture of retinal ganglion cells and amacrine cells from adult mice

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures...

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Published in:PloS one 2020-12, Vol.15 (12), p.e0242426
Main Authors: Park, Yong H, Snook, Joshua D, Zhuang, Iris, Shen, Guofu, Frankfort, Benjamin J
Format: Article
Language:English
Subjects:
Aging
Amacrine cells
Amacrine Cells - drug effects
Amacrine Cells - physiology
Animals
Antibodies
Axonogenesis
Biology and Life Sciences
Brain-derived neurotrophic factor
CD57 antigen
Cell culture
Cell survival
Cell Survival - drug effects
Cell Survival - physiology
Cells, Cultured
Cloning
Culture media
Development and progression
Diagnosis
Dose-Response Relationship, Drug
Embryos
Female
Health aspects
In vivo methods and tests
Inhibitory Concentration 50
Inhibitory postsynaptic potentials
Laboratories
Male
Medical schools
Medicine
Medicine and Health Sciences
Methods
Mice
Molecular biology
Neonates
Nervous system
Nervous system diseases
Neurological diseases
Neuronal Outgrowth
Neurons
Optimization
Physiological aspects
Pluripotency
Population studies
Populations
Primary Cell Culture - methods
Puromycin
Puromycin - pharmacology
Research and Analysis Methods
Retina
Retinal ganglion cells
Retinal Ganglion Cells - drug effects
Retinal Ganglion Cells - physiology
Stem cell transplantation
Stem cells
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cited_by cdi_FETCH-LOGICAL-c692t-9834c8f6e78b7c79caceee4e07d36bda3d6d1ba9db4c9a96796c60e17e813e1f3
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container_issue 12
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container_title PloS one
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creator Park, Yong H
Snook, Joshua D
Zhuang, Iris
Shen, Guofu
Frankfort, Benjamin J
description Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500μm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.
doi_str_mv 10.1371/journal.pone.0242426
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Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500μm in length. 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Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33284815</pmid><doi>10.1371/journal.pone.0242426</doi><tpages>e0242426</tpages><orcidid>https://orcid.org/0000-0001-8096-9812</orcidid><orcidid>https://orcid.org/0000-0001-7343-5652</orcidid><oa>free_for_read</oa></addata></record>
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source PubMed (Medline); ProQuest - Publicly Available Content Database
subjects Aging
Amacrine cells
Amacrine Cells - drug effects
Amacrine Cells - physiology
Animals
Antibodies
Axonogenesis
Biology and Life Sciences
Brain-derived neurotrophic factor
CD57 antigen
Cell culture
Cell survival
Cell Survival - drug effects
Cell Survival - physiology
Cells, Cultured
Cloning
Culture media
Development and progression
Diagnosis
Dose-Response Relationship, Drug
Embryos
Female
Health aspects
In vivo methods and tests
Inhibitory Concentration 50
Inhibitory postsynaptic potentials
Laboratories
Male
Medical schools
Medicine
Medicine and Health Sciences
Methods
Mice
Molecular biology
Neonates
Nervous system
Nervous system diseases
Neurological diseases
Neuronal Outgrowth
Neurons
Optimization
Physiological aspects
Pluripotency
Population studies
Populations
Primary Cell Culture - methods
Puromycin
Puromycin - pharmacology
Research and Analysis Methods
Retina
Retinal ganglion cells
Retinal Ganglion Cells - drug effects
Retinal Ganglion Cells - physiology
Stem cell transplantation
Stem cells
title Optimized culture of retinal ganglion cells and amacrine cells from adult mice
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