Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Toegl, L.
Organotypic slice cultures of the adult rat brain
An approach for improved cell viability
Humanmedizin; [ Diplomarbeit ] Medizinische Universität Graz; 2023. pp. 81
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Patz Silke
- Ücal Muammer
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- Abstract:
- Introduction: Organotypic slice culture of the brain (OTC) is a valuable technique in neuroscience. OTC slices are commonly tissues from neonatal donor animals. In adult slices, cell viability continuously drops - reason for the lack of its widespread application to studies on adult brain pathologies. Hence, improvement of viability is desirable. Reports point to potential benefits of non-classical, serum-free culture medium formulations (Neurobasal A, NB-A), insulin and ascorbic acid (AA) in adult OTC. The aim of this study was to assess if NB-A, the supplementation of insulin, AA or the combination of the last two can better retain live cells in whole-brain OTC or in cortical, hippocampal, or thalamic tissue therein. Methods: Adult whole-brain slice cultures prepared from 9–12-week-old rats were treated with 1) a serum-containing medium (BME), 2) BME + insulin, 3) BME + AA, 4) BME + insulin + AA, 5) NB-A, 6) NB-A + insulin, 7) NB-A + AA, 8) NB-A + insulin + AA. On days 1, 3, 5 and 7 in-vitro, after labelling with calcein (live cells) and EthD-1 (dead cells), confocal laser scanning microscopy images were taken, and cells counted by BoneJ plugin in FIJI image processing software in cortex, hippocampus, and thalamus. A total cell count was calculated. Experimental groups and dead control were compared by one-way ANOVA or Kruskal-Wallis’ test. Results: Neurobasal A medium was significantly better than BME on day 3 in total cell counts and cortex. Generally, groups treated with insulin, AA or both did not significantly differ from those without them, but all supplements could be beneficial for certain tissues at certain timepoints. Insulin + AA supplement had distinct effects other than insulin or AA. Discussion: The investigated media and supplements are interesting for further investigation but might require additional measures to improve viability. Tailoring highly specific media optimized to brain region and time point of interest or considering other environmental factors is advisable. In this study, the following important limitations apply: Calcein/EthD-1 staining cannot discriminate between cell types, sample size differs between groups and is small. Double-staining and challenges in digital cell location occurred.