Live Imaging of Primary Neurons in Long-Term Cryopreserved Human Nerve Tissue
| Autor: | Ingrid Demedts, Tim Vanuytsel, Piyush Jain, Marina Fortea, Carla Cirillo, Pieter Vanden Berghe, Jan Tack |
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| Rok vydání: | 2021 |
| Předmět: |
human primary neuron
Biology cryopreservation MOUSE Serotonergic Novel Tools and Methods Cryopreservation enteric nervous system life imaging Live cell imaging Humans Nerve Tissue Neurons Science & Technology General Neuroscience Endoscopic biopsy Neurosciences Functional measurement Histology General Medicine Research Article: Methods/New Tools CELLS BIOPSIES Immunohistochemistry Enteric nervous system Calcium Neurosciences & Neurology Life Sciences & Biomedicine Neuroscience |
| Zdroj: | eNeuro |
| ISSN: | 2373-2822 |
| Popis: | 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 (Ca2+) 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. Significance Statement We describe a novel protocol for live imaging of human primary neurons after long-term cryopreservation. This provides a meaningful advance in neuroscience, as it enables sharing live tissues between research centers for a variety of experiments. As such, this technique will broaden research networks and facilitate collaborations between institutes worldwide contributing with different expertise. |
| Databáze: | OpenAIRE |
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