Name: organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells
Uploaded: 2017-08-27T13:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

The authors would like to thank Christine Auste for her support with the video recording and Chalid Ghadban for his excellent technical assistance. Urszula Grabiec was supported by the Roux Programm FKZ 29/18.

Animal experiments were performed in accordance with the Policy of Ethics and the Policy on the Use of Animals in Neuroscience Research as approved by the European Communities Council Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes.
1. Preparation of Instruments and Culture Media
<ol>
	<li>For preparation of OHSC use the following set of instruments: two small scissors, two curved tweezers, one tweezer with a fine tip, three blades (two of size 11, one of size 15), three scalpel holders, round filter papers (diameter: 35 mm), agar, one razor blade, one unmodified Pasteur pipette, and one modified Pasteur pipette without a tip. Sterilize all materials in an autoclave before usage (Figure 1).</li>
	<li>Weigh 5 g agar and dissolve it in 100 mL distilled water. Sterilize the solution for 20 min at 121.7 &#176;C at 210.8 kPa in an autoclave. Distribute the 3 mL liquid agar solution in 60-mm Petri dishes using a sterile glass pipette, allow it to solidify for 5 h, cover with plastic paraffin film to avoid contamination and store at 4 &#176;C until further use. Agar blocks are needed to stabilize the brains during the slicing procedure.</li>
	<li>Media
	<ol>
		<li>Make 200 mL of the preparation medium (pH 7.35) consisting of 198 mL minimal essential medium (MEM) and 2 mL L-glutamine solution (final concentration 2 mM). Prepare the solution on the day of media preparation and store it at 4 &#176;C.</li>
		<li>Prepare 100 mL of the culture medium consisting of 49 mL MEM, 25 mL Hanks&#39; balanced salt solutions (HBSS), 25 mL (v/v) normal horse serum (NHS), 1 mL L-glutamine solution (final concentration 2 mM), 100 &#181;g insulin, 120 mg glucose, 10 mg streptomycin, 10,000 U penicillin, and 800 &#181;g vitamin C as reported previously. Warm the medium (37 &#176;C), adjust the pH value to pH 7.4 and sterile filter (0.2 &#181;m pore size). Repeat the procedure (warming up, pH adjustment, etc.) every second day before changing the medium. Use the medium at the most for one week when stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Fill one 35-mm Petri dish with preparation medium to store the brain. Place two empty Petri dishes for collecting the tissue on a cooling pack in the working area.</li>
</ol>
2. Preparation and Slicing with a Vibratome
<ol>
	<li>Use brains from 7-9 day old rats or 4-5 day old mice for the OHSC preparation according to Stoppini et al.4 After decapitation of animals, remove the skin from the skull with scissors.</li>
	<li>Introduce the blade of a fine scissor into the foramen magnum and open the skull by cutting along the caudal (back) rostral (front) axis. Make two cuts perpendicular to the first one, so that the scissors point towards the left and right ear, respectively (&#34;T-incision&#34;).</li>
	<li>Open the skull carefully with fine forceps, paying attention not to injure the brain. Use a scalpel (blade size 11) to cut the most rostral part of the frontal pole and the cerebellum.</li>
	<li>By means of a spatula, remove the brain and place it carefully in the Petri dish filled with preparation medium (Figure 2). Position the brain on the specimen holder and fix it with medical cyanoacrylate glue. Use the pieces of agar to assure mechanical stabilization.</li>
	<li>Dissect the tissue horizontally in 350 &#181;m thick OHSC using a sliding vibratome.</li>
	<li>Evaluate the slices optically using a binocular microscope. Discard OHSC of low quality immediately. It is important to take only those slices with intact cytoarchitecture isolated from the middle part of the hippocampus (see Figures 3 and 4) between the dorsal and the ventral hippocampus.</li>
	<li>Separate the hippocampal region and the entorhinal cortex using a scalpel (round blade size 15; Figure 3). The perforant pathway and entorhinal cortex must be preserved.</li>
	<li>Six to eight OHSC are obtained from each brain. Transfer 2-3 slices into one cell culture insert (pore size 0.4 &#181;m) and place it in one well of a 6-well culture dish containing 1 mL culture medium per well.</li>
	<li>Incubate the 6-well dishes at 35 &#176;C in a fully humidified atmosphere with 5% (v/v) CO2 and change the cell culture medium every second day. 
	NOTE: Conduct your experiments at 6 day in vitro (div). Inflammatory reactions associated with the slicing procedure disappear by day 6. At this stage, microglial cells show a ramified morphology again and synaptic connections have matured.</li>
</ol>
3. Evaluation of Tissue Quality
<ol>
	<li>Fixation, labeling and visualization of degenerating neurons in OHSC
	<ol>
		<li>After performing the experiments, incubate the OHSC with 5 &#181;g/mL propidium iodide (PI) for 2 h prior to fixation, in order to stain the nuclei of degenerating neurons. 
		CAUTION: PI is a suspected carcinogen, always wear personal protective equipment (PPE) such as gloves.</li>
		<li>Wash the OHSC with 0.1 M phosphate buffer (pH 7.4) and fix them with a 4% (v/v) solution of paraformaldehyde (PFA) for 24 h. 
		CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.</li>
		<li>Wash the OHSC in inserts with 1 mL PBS and use a rigger brush to separate slices from the membrane.</li>
		<li>Label the OHSC with IB4 dye in a 24-well plate (Figure 5).</li>
	</ol>
	</li>
	<li>Conduction of tumor invasion experiments using fluorescently labeled single cells
	<ol>
		<li>24 h prior to the start of an experiment, label the tumor cells by using the fluorescent dye Carboxyfluorescein diacetate succinimidyl ester (CFDA SE).</li>
		<li>Detach and count the tumor cells using a Neubauer chamber.</li>
		<li>Resuspend the cells in medium, such that 10 &#181;L of suspension contains the desired cell number (normally 50,000 or 100,000 cells).</li>
		<li>Apply 10 &#181;L of the cell suspension onto the slice culture and allow the cells to invade for 3 days.</li>
		<li>At the end of the experiment, fix the slice cultures using 4% (v/v) PFA for 24 h, and label the co-cultures with PI for another 24 h to visualize the cytoarchitecture. 
		CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.</li>
		<li>Mount the co-cultures onto a cover slip for further analysis using the mounting media.</li>
	</ol>
	</li>
</ol>
4. Evaluation of OHSC Experiments
Analyze the OHSC with a confocal laser scanning microscope (CLSM). For detection of PI labeled, degenerating neurons or the PI labeled cytoarchitecture use monochromatic light of the wavelength &#955; = 543 nm and an emission band pass filter for wavelengths &#955; = 585-615 nm. For CFDA labeled tumor cells or IB4 microglia, use an excitation wavelength of &#955; = 488 nm. For both experimental types, record a z-stack with 2 &#181;m thick optical slices and used for evaluation.

The authors have nothing to disclose

The present protocol describes the preparation of OHSC. This model allows testing of intrinsic capabilities and reactions of brain tissue after the application of physiological and pathological stimuli. Besides analyses of electrophysiological parameters, OHSC can be lesioned and the effects of damage on all cell types can be determined. Treatment with different substances and the detailed description of lesioning processes or healing in the absence of macrophages and lymphocytes is possible.
...

OHSC are a well-characterized in vitro model to study both physiological and pathological properties of neurons, astrocytes and microglia1. It is easy to control the extracellular environment and monitor the cellular and morphological changes after various stimuli. The organization of hippocampal neurons and their connections are well preserved after preparation2,3. Out of several advantages, OHSC allow monitoring of brain injury and tumor invasion without animal surgery. Six to eight OHSC can be obtained from a single rodent brain. OHSC therefore help to significantly reduce the number of animals and allow testing multiple drug concentrations, genetic manipulations or different lesion models in the same animal. In slice-based assays, experimental conditions can be precisely controlled. Additionally, time dependent development of pathological conditions like secondary damages can easily be monitored by time-lapse imaging.
In the given protocol, originally established by Stoppini et al.4, the preparation steps are described and important morphological landmarks for the selection of appropriate slices are highlighted. We recommend the preparation of postnatal day 7-9 rats or postnatal day 4-5 mice. In these periods, OHSC show a robust resistance to mechanical traumas and a high potential for reorganization of neuronal circuits. In contrast, preparations from embryonic or adult rats rapidly change their structure and lose their organotypic morphology during cultivation and are therefore less suitable for studying long-term processes in basic research5,6,7,8,9,10,11. Another critical point for the survival rate of OHSC is the thickness of the slice itself as the diffusion and thus nutrient supply are limited12,13,14.

<table><tbody><tr><td>6-Well</td><td>Falcon</td><td>35-3046</td><td></td></tr><tr><td>Agar</td><td>Fluka</td><td>5040</td><td></td></tr><tr><td>Autoclav</td><td>Systec</td><td>DX-45</td><td></td></tr><tr><td>CFDA</td><td>&amp;nbsp;Thermo Fisher</td><td>V12883</td><td></td></tr><tr><td>Confocal laser scanning microscope (CLSM) LSM700</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Eagle&amp;acute;s Minimal Essential Medium&amp;nbsp;</td><td>Invitrogen</td><td>32360-034</td><td></td></tr><tr><td>Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I</td><td>Vector Labs</td><td>FL-1101</td><td></td></tr><tr><td>Glucose</td><td>Merk</td><td>1083371000</td><td></td></tr><tr><td>Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>24020-133</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (without Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>14170-138</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>I5500</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td></td></tr><tr><td>L-Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>LN229</td><td>Cell-Lines-Service</td><td>300363</td><td></td></tr><tr><td>Medical cyanoacrylate glue (Histoacryl glue)</td><td>B.Braun</td><td>1050052</td><td></td></tr><tr><td>Millicell Culture Inserts</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>NMDA N-methyl-D-aspartic acid</td><td>Sigma Aldrich</td><td>M3262</td><td></td></tr><tr><td>Normal Horse Seum</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Petri dishes (all sizes)</td><td>Greiner</td><td>627160/664160/628160</td><td></td></tr><tr><td>PFA</td><td>Roth</td><td>0335.1</td><td>toxic</td></tr><tr><td>Propidium iodid (PI)</td><td>Sigma Aldrich</td><td>81845-25MG</td><td>toxic</td></tr><tr><td>U138</td><td>ATCC</td><td>HTB-14</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>Leica VT 1200</td><td></td></tr></tbody></table>

organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Neuroprotection studies: To determine neuronal damage, the number of PI positive nuclei and IB4 positive microglia in every third optical section of the granule cell layer (GCL) of the dentate gyrus (DG) was counted. For tumor invasion experiments, the maximal intensity z-projection of the stack was used for calculating the area covered by tumor cells, as a measure of invasion and to visualize different invasion patterns (Figure 6)...

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

organotypic-hippocampal-slice-cultures-as-model-to-study

Research

JoVE Journal

Neuroscience

12.8K Views.  Martin Luther University Halle-Wittenberg. Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Video: Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicine

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://app.jove.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...