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Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue

Tissue cryopreservation provides a convenient solution for tackling one of the major problems in neuroscience research, namely, the scarce availability of human nerve tissues, especially if needed alive. While brain tissue can be used only postmortem, live nerve tissue can reasonably well be harvest...

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Detalles Bibliográficos
Autores principales: Fortea, Marina, Jain, Piyush, Demedts, Ingrid, Tack, Jan, Vanuytsel, Tim, Cirillo, Carla, Vanden Berghe, Pieter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Society for Neuroscience 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8638677/
https://www.ncbi.nlm.nih.gov/pubmed/34759050
http://dx.doi.org/10.1523/ENEURO.0388-21.2021
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author Fortea, Marina
Jain, Piyush
Demedts, Ingrid
Tack, Jan
Vanuytsel, Tim
Cirillo, Carla
Vanden Berghe, Pieter
author_facet Fortea, Marina
Jain, Piyush
Demedts, Ingrid
Tack, Jan
Vanuytsel, Tim
Cirillo, Carla
Vanden Berghe, Pieter
author_sort Fortea, Marina
collection PubMed
description Tissue cryopreservation provides a convenient solution for tackling one of the major problems in neuroscience research, namely, the scarce availability of human nerve tissues, especially if needed alive. While brain tissue can be used only postmortem, live nerve tissue can reasonably well be harvested from the periphery. A valuable source of primary neurons is the intestine, which compared with brain has the advantage to be safely accessible via endoscopy. The nerve tissue innervating the intestine (the enteric nervous system; ENS) can be sampled with regular endoscopic biopsy forceps and remains viable for multiple physiological and immunohistochemical tests, as previously demonstrated. Here, we present a method to preserve, over longer periods of time, human primary neurons contained in these biopsies. The use of a cryoprotective agent and the application of controlled cooling revealed to be crucial to properly store the nerve tissue and to enable functional measurements after thawing. These primary neurons were evaluated for functionality (live imaging) and morphology (histology) up to one year after cryopreservation. Calcium (Ca(2+)) imaging indicated that human primary neurons remained viable and responded to selective stimulations (serotonergic and nicotinic agonists) after cryopreservation. Additionally, immunohistochemistry performed with specific neuronal markers showed that nerve structure and neuronal morphology were retained, with no signs of cellular damage. In this study, we demonstrate that the human ENS is a realistic source of primary neurons, which can be successfully preserved over long times and as such can be exploited both for gastrointestinal-specific as well as for general neuroscience research.
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spelling pubmed-86386772021-12-03 Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue Fortea, Marina Jain, Piyush Demedts, Ingrid Tack, Jan Vanuytsel, Tim Cirillo, Carla Vanden Berghe, Pieter eNeuro Research Article: Methods/New Tools Tissue cryopreservation provides a convenient solution for tackling one of the major problems in neuroscience research, namely, the scarce availability of human nerve tissues, especially if needed alive. While brain tissue can be used only postmortem, live nerve tissue can reasonably well be harvested from the periphery. A valuable source of primary neurons is the intestine, which compared with brain has the advantage to be safely accessible via endoscopy. The nerve tissue innervating the intestine (the enteric nervous system; ENS) can be sampled with regular endoscopic biopsy forceps and remains viable for multiple physiological and immunohistochemical tests, as previously demonstrated. Here, we present a method to preserve, over longer periods of time, human primary neurons contained in these biopsies. The use of a cryoprotective agent and the application of controlled cooling revealed to be crucial to properly store the nerve tissue and to enable functional measurements after thawing. These primary neurons were evaluated for functionality (live imaging) and morphology (histology) up to one year after cryopreservation. Calcium (Ca(2+)) imaging indicated that human primary neurons remained viable and responded to selective stimulations (serotonergic and nicotinic agonists) after cryopreservation. Additionally, immunohistochemistry performed with specific neuronal markers showed that nerve structure and neuronal morphology were retained, with no signs of cellular damage. In this study, we demonstrate that the human ENS is a realistic source of primary neurons, which can be successfully preserved over long times and as such can be exploited both for gastrointestinal-specific as well as for general neuroscience research. Society for Neuroscience 2021-11-30 /pmc/articles/PMC8638677/ /pubmed/34759050 http://dx.doi.org/10.1523/ENEURO.0388-21.2021 Text en Copyright © 2021 Fortea et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
spellingShingle Research Article: Methods/New Tools
Fortea, Marina
Jain, Piyush
Demedts, Ingrid
Tack, Jan
Vanuytsel, Tim
Cirillo, Carla
Vanden Berghe, Pieter
Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
title Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
title_full Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
title_fullStr Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
title_full_unstemmed Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
title_short Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
title_sort live imaging of primary neurons in long-term cryopreserved human nerve tissue
topic Research Article: Methods/New Tools
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8638677/
https://www.ncbi.nlm.nih.gov/pubmed/34759050
http://dx.doi.org/10.1523/ENEURO.0388-21.2021
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