研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

Preparation and slicing with a vibratome. Three agar blocks are needed to stabilize the brain during the slicing procedure. To avoid contamination, use an Autoclave set of instruments.

Remove the agar. Should be dried on the filter paper before gluing it onto the metal plate with medical Cyanoacrylate glue. By means of a spatula, remove the brain and place it carefully in the Petri dish filled with preparation medium.

Next, shortly dry the brain on the filter paper. Cover the metal plate with medical Cyanoacrylate glue. Use the tweezers and the spatula to position the brain on the specimen holder.

Use the previously prepared two pieces of agar to ensure mechanical stability from both sides. At this point, the brain should be placed next to the agar block and stabilized with two further blocks to prevent damage during the slicing. Place the metal plate in the vibratome and cover the tissue with cold preparation medium.

Thereafter, dissect the tissue horizontally in 350 micrometers thick OHSC using a sliding vibratome. Transfer the slices to the Petri dish, using the back of a broken Pasteur pipette to collect the slices. Keep brain slices in cold preparation medium.

Evaluate the slices optically with a binocular microscope. Separate the hippocampal region and enter inner cortex using a scalpel with a round blade of size 15. It is important to take only those slices within intact cytoarchitecture isolated from the middle part of the hippocampus between the dorsal and the ventral hippocampus.

OHSCs of low quality should be discarded immediately. Transfer two to three of the obtained slices into cell culture insert with a pore size of 0.4 micrometer. Aspirate a preparation medium from the top of the cell culture insert using a Pasteur pipette.

Place the insert in a six well culture dish containing one millimeter culture medium per well. Incubate the six well dishes at 35 degrees Celsius in fully humidified atmosphere with 5%CO2 and change the cell culture medium every second day. After six days in culture for rat tissue, or 14 days for murine tissue, inflammatory reactions associated with the slicing procedure itself have been disappeared.

Experiments can now be conducted. Representative results for neuroprotection studies. Image part A shows unlesioned OHSC kept in culture medium for nine days in vitro without any treatment serving as control slices.

As confirmed by confocal lasers scanning microscopy, control OHSC showed good neuronal preservation. The Isolectin B4 positive microglial cells were mainly localized in the molecular and plexiform layers and exhibited a ramified phenotype. In the granular cell layer of the dentate gyrus, only a very few Isolectin B4 positive microglial cells and virtually no Propidium Iodide positive nuclei were found.

Another set of OHSC, shown in image part B, were kept in culture medium for six days, lesioned with 50 micromolar NMDA for four hours. And kept in culture medium for another three days. After NMDA lesion, a massive accumulation of activated Isolectin B4 positive microglial cells adhered to the site of neuronal injury, throughout all layers of the dentate gyrus.

Quantitative analysis revealed that in granular cell layer of the dentate gyrus, the number of Isolectin B4 positive microglial cells was higher in lesioned OHSC compared to the control conditions. In a similar manner, the number of Propidium Iodide positive degenerating neurons were strongly increased in the granular cell layer of NMDA lesioned slices. Sample results for tumor invasion studies.

All images depict results of tumor invasion SS using OHSC that were imaged with a confocal laser scanning microscope. Red color labels the cytoarchitecture of the OHSC and green tumor cells. Image A shows a typical invasion pattern for U138 glioblastoma cells that were allowed to invade for four days.

Clear, spherical, distinguished tumors can be observed and the OHSC structure seems to be intact with a clearly visible dentate gyrus and Cornu Ammonis. Image B shows a different invasion pattern, generated by LN229 glioblastoma cells after four days. In this case, single tumors cannot be clearly distinguished due to the formed network that is covering most of the OHSC, even though the cytoarchitecture remains intact with an undeformed dentate gyrus and Cornu Ammonis.

It can furthermore be concluded that LN229 cells are more invasive than U138 cells. Image C and D show the same tumor invasion result for LN229 cells after four days of invasion. On the left side, it can be observed that the tumor mass is covering almost the whole field of view.

Image D shows the corresponding Propidium Iodide dyeing. In this slide, the cytoarchitecture is no longer intact as the Cornu Ammonis is not visible anymore and the dentate gyrus is distorted as well. A possible reason for such a result might be an OHSC that was not fully intact at the beginning of that experiment, a too high number of applied tumor cells, or a too long invasion time that resulted in the observed loss of structure.

Thus, these kind of invasion results should be discarded. Organotypic hippocampal slice cultures represent a very good in vitro model reflecting the physiology of the brain with intact neuronal connections. As of one model, several slice cultures can be obtained, it allows to test multiple conditions in tissue from one animal and the results are highly reproducible.

Due to the high degree of accessibility, organotypic hippocampal slice cultures are excellently suited for a multitude of experimental types. This includes electrophysiological and neurogenesis studies, but also experiments on neuroprotection, tumor invasion, and interactions of tumor cells with healthy brain tissue.