Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We thank Sean McDermott for his technical assistance and support, and Maya Epstein for the hand-drawn illustrations shown in Figure 1.

All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.
1. Preparation prior to organotypic cerebellar slice cultures
<ol>
	<li>In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture medium in a 250 mL bottle-top vacuum filter attached to a sterile bottle receiver. Add 100 mL of Basal Medium Eagle (BME), 50 mL of Hanks&#39; Balanced Salt Solution (HBSS), 50 mL of heat-inactivated horse serum, 1 mL of 200 mM L-Glutamine (final concentration 1 mM), and 5 mL of 200 g/L glucose (final concentration 5 mg/mL). Vacuum-filter the culture medium and keep it at +4 &#176;C for up to a month.</li>
	<li>In the sterilized biosafety cabinet, take out a 6-well culture plate from its package. Fill each well with 1 mL of culture medium. If a treatment is tested, add the pharmacological agent to the treated well, and the same volume of vehicle solution for the control well. In the present study, we added 1 &#181;L of sterile 3 M KCl solution to the treated well (30 mM final) and 1 &#181;L of sterile water to the control wells.</li>
	<li>Bring inside the cell culture hood the needed number of individually wrapped cell culture inserts with hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size = 0.4 &#181;m). Carefully unwrap them and take out the cell culture inserts with sterile forceps. Drop them one by one in a well of a 6-well plate, avoiding bubbles at the interface between the insert membrane and the cell culture medium. Let the inserts equilibrate in the medium in a 37 &#176;C, 5% CO2 incubator for at least 2 h before culture. 
	NOTE: The tissue chopper permanently resides in the cell culture hood.</li>
	<li>Prepare two 200 mL beakers, one filled with 150 mL sterile water, and the other filled with 150 mL 70% ethanol. Dip the surgical tools in the 70% ethanol bath and then in water prior to using them. 
	NOTE: Do not use surgical tools immediately following contact with ethanol.</li>
	<li>Disinfect the double-edged blade in the 70% ethanol beaker. Place it on the blade holder using sterile forceps and hold it into place using the blade clamp wrench. Let it dry until use.</li>
	<li>Disinfect the plastic disc provided with the tissue chopper in the 70% ethanol beaker. Spray the cutting table with 70% ethanol prior to placing the disc on it. Let dry until use.</li>
</ol>
2. Dissection and Cerebellar Organotypic Slice Cultures
<ol>
	<li>Bring the mouse pup inside the cell culture hood to perform the dissection.</li>
	<li>Decapitate the pup quickly using straight operating scissors (Figure 1A).</li>
	<li>While holding the pup&#39;s head by the nose with straight dressing forceps, cut open the scalp using straight eye scissors, starting from the posterior end, and going lateral to midline.</li>
	<li>Proceed identically for the skull. 
	NOTE: Make sure to point scissor tips outwards to avoid damaging the cerebellum during dissection.</li>
	<li>Take out the brain and transfer it to a 60-mm dish filled with cold HBSS + 5 mg/mL glucose.</li>
	<li>Carefully dissect out the cerebellum using sterile straight fine forceps (Figure 1B). Transfer it onto the plastic disc placed on the cutting table of the tissue chopper using sterile curved fine forceps.</li>
	<li>Orient the tissue by rotating the cutting table to perform parasagittal sections.</li>
	<li>Move the cutting table by pulling the table release knob to the right, so the blade is positioned at the edge of the tissue.</li>
	<li>Adjust the slice thickness to 350 &#181;m and the blade speed to medium.</li>
	<li>Start the chopper. Once the whole cerebellum has been sliced (Figure 1C), turn off the chopper.</li>
	<li>Remove the plastic disc with sterile forceps.</li>
	<li>Carefully bring the plastic disc close to a 60-mm dish containing HBSS + 5 mg/mL glucose. Pipette cold HBSS + 5 mg/mL glucose onto the tissue using a sterile transfer pipette to allow harvesting of cerebellar slices in the buffer-filled dish.</li>
	<li>Separate the cerebellar slices from each other using a microprobe. Take out good quality sections with the transfer pipette (it is recommended to use tissue sections that are close to the vermis) and drop them off in a pre-equilibrated cell culture insert (Figure 1D).</li>
	<li>Position the cerebellar slices on the cell culture insert using the microprobe, then carefully remove the excess HBSS + 5 mg/mL glucose solution with the transfer pipette. 
	NOTE: At this stage the tissue is extremely tender and prone to physical damage. The maximum number of cerebellar slices per cell culture insert depends on the age of the donor animal. 6&#8211;8 slices can be cultured for a P0&#8211;P4-old animal, and 4&#8211;6 slices for animals older than P5.</li>
	<li>Place the culture dish in the incubator, changing the medium completely every 2&#8211;3 days.</li>
	<li>To assess Purkinje cell survival, slices can be collected as early as 5 days in vitro. For other purposes, they can be maintained in culture for several weeks (Figure 1F). 
	NOTE: In the case of Purkinje cell survival experiments, there is no need to renew the pharmacological treatment when changing cell culture medium (Figure 1F).</li>
</ol>
3. Immunofluorescence
<ol>
	<li>Take out the 6-well plate from the incubator. Remove cell culture medium, wash the cell culture insert with 1x PBS, and fix the slices with cold 4% paraformaldehyde solution for 1 h, with 1 mL under the cell culture insert and 500 &#181;L on top of the cell culture insert.</li>
	<li>Wash the inserts 4x 10 min with 1x PBS, with 1 mL under and 500 &#181;L on top of the insert, on an orbital shaker.</li>
	<li>With a brush, transfer the cerebellar slices from 1 cell culture insert to 1 well of a 24-well pre-filled with 1x PBS, 0.25% Triton X-100, and 3% BSA (PBS-TB), 500 &#181;L/well.</li>
	<li>Permeabilize and block the slices by incubation in PBS-TB for 1 h at room temperature.</li>
	<li>Incubate with primary antibodies diluted in the PBS-TB solution, overnight at + 4 &#176;C, on an orbital shaker, 200 &#181;L/well.</li>
	<li>On the following day, perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 &#181;L/well.</li>
	<li>Incubate with secondary antibodies diluted in the PBS-TB solution, two hours at room temperature, on an orbital shaker, and protected from light, 200 &#181;L/well.</li>
	<li>Perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 &#181;L/well.</li>
	<li>Counterstain the slices using Hoechst 33342, diluted in 1x PBS (2 &#181;g/mL), for 10 min at room temperature, protected from light, 500 &#181;L/well.</li>
	<li>Mount the cerebellar slices on a microscopy slide with a transfer pipette. Let them air-dry completely, protected from light. Re-humidify them with 1x PBS and apply a coverslip covered with few drops of mounting medium (~80 &#181;L). Avoid making any bubbles.</li>
	<li>Once the mounting medium has cured, cerebellar sections are ready to be imaged.</li>
</ol>
4. Imaging and Quantification of Cell Survival
<ol>
	<li>Turn on the microscope and lasers according to the manufacturer and/or the imaging core facility&#39;s instructions.</li>
	<li>Start the acquisition software.</li>
	<li>Using a 10X objective, locate non-altered cerebellar slices that present 9&#8211;10 lobules (usually cerebellar sections close to the vermis).</li>
	<li>Activate z-stack acquisition. Define the range of the z-stack to include all Purkinje cells present in the cerebellar slice. Set a 2 &#181;m step size and start the acquisition. Save the acquired picture in the format of the acquisition software manufacturer to preserve the associated metadata.</li>
	<li>Open the acquired file in ImageJ using the Bio-formats plugin18.</li>
	<li>Go to Image &#62; Stacks &#62; Z-project&#8230; (Figure 2A).</li>
	<li>Choose Max Intensity in the Projection type dropdown menu and click OK (Figure 2B).</li>
	<li>A flattened 2-D image will be generated. Double click on the Multi-point tool to let the Point Tool window appear. Uncheck the Label points option to ease counted cells visualization. Then, click directly on a soma of a Purkinje cell to begin counting (Figure 1E). The total number of counted cells will be indicated at the bottom of the Point Tool window (Figure 3). 
	NOTE: Usually, at least 3 non-damaged sections containing 9&#8211;10 lobules per cell culture insert can be quantified. To assess the neuroprotective effect of a pharmacological agent, at least 3 independent experiments should be performed.</li>
</ol>

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The authors have nothing to disclose.

Cerebellar slice culture is a powerful tool to study programmed Purkinje cell death during postnatal development. This technique can be used to rapidly screen candidate molecules for their neuroprotective potential. The main advantage is that the setup is simple and very cost effective, and only requires a modest investment in equipment (a vibratome can cost up to 3 times more than a tissue chopper). Moreover, 10 to 15 healthy slices can be generated from one mouse pup, allowing for different assays to be performed in pa...

In vitro modeling is an essential tool in biomedical research. It allows investigators to study and tightly control specific mechanisms in restricted cell types, or in isolated systems/organs. Organotypic slice culture is a widely used in vitro technique, especially in the field of neuroscience1. The method was first established by G&#228;hwiler, who cultured brain slices using the roller tube technique2, and later modified by Yamamoto et al., who introduced the use of a microporous membrane to perform cortical slice cultures3. Compared to primary cell cultures, organotypic slice cultures present the advantage of preserving the cytoarchitecture of the tissue, as well as native cell-cell connections in the plane of the tissue section.
Organotypic slices have been cultured from many parts of the central nervous system, such as the hippocampus4, cortex5, striatum6, cerebellum4,7, and spinal cord8,9, among others. They have been proven to be a powerful tool in drug discovery studies10. The effects of neuroactive molecules can be assessed in many ways: survival and neurodegeneration using immunostaining and biochemistry assays, neuronal circuit formation, or disruption using electrophysiology and live-imaging.
The goal of this work is to describe a simple method to perform organotypic cerebellar slice culture, which is known to be a relevant model to mimic cerebellar development in vitro. Particularly, we focused on the study of Purkinje cell developmental death. In vivo, Purkinje cells undergo apoptosis during the first postnatal week, peaking at postnatal day 3 (P3)11. The same pattern is observed in cerebellar slice culture, with Purkinje neurons dying by apoptosis when cerebella are taken from animals between P1 and P8, with a peak at P34,12. The use of the organotypic cerebellar slice cultures has allowed to identify several neuroprotective molecules7,13, as well as understanding part of the mechanisms involved in this programmed cell death14,15,16. Here, we describe a protocol based upon the study of Stoppini et al.17 in hippocampus, and adapted to cerebellum by Dusart et al.4 It includes rapid dissection and chopping of postnatal cerebella; slicing culture onto a cell culture insert containing a microporous membrane, with or without neuroprotective treatment; and immunofluorescence staining to assess neuronal survival.

<table><tbody><tr><td>Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody</td><td>ThermoFisher scientific</td><td>A21203</td><td></td></tr><tr><td>Basal Medium Eagle (BME)</td><td>ThermoFisher scientific</td><td>21010046</td><td></td></tr><tr><td>Biosafety cabinet Class II, Type A2</td><td>NuAire</td><td>NU-540-400</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Millipore Sigma</td><td>A2153</td><td></td></tr><tr><td>Brush</td><td></td><td></td><td></td></tr><tr><td>anti-Calbindin D-28K antibody (CB-955)</td><td>Abcam</td><td>ab82812</td><td></td></tr><tr><td>CO2 Incubator</td><td>NuAire</td><td>NU-5700</td><td></td></tr><tr><td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td><td>Fisher Scientific</td><td>07-200-83</td><td></td></tr><tr><td>Coverslips, 22 x 50 mm</td><td>Fisher Scientific</td><td>12-545-E</td><td></td></tr><tr><td>Dressing forceps, straight</td><td>Harvard Apparatus</td><td>72-8949</td><td></td></tr><tr><td>Double edge blades</td><td>Fisher Scientific</td><td>50949411</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon Labs, Inc</td><td>2701</td><td></td></tr><tr><td>Eye Scissors, straight</td><td>Harvard Apparatus</td><td>72-8428</td><td></td></tr><tr><td>Fine forceps</td><td>Fisher Scientific</td><td>16-100-127</td><td></td></tr><tr><td>L-Glutamine 100X</td><td>ThermoFisher scientific</td><td>25030149</td><td></td></tr><tr><td>Glucose solution</td><td>ThermoFisher scientific</td><td>A2494001</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt Solution</td><td>ThermoFisher scientific</td><td>14025092</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate</td><td>Fisher Scientific</td><td>H21492</td><td></td></tr><tr><td>Horse Serum, heat inactivated, New Zealand origin</td><td>ThermoFisher scientific</td><td>26050088</td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>Fisher Scientific</td><td>NC9914528</td><td></td></tr><tr><td>Microprobes</td><td>Fisher Scientific</td><td>08-850</td><td></td></tr><tr><td>Millicell Cell Culture Inserts</td><td>Millipore Sigma</td><td>PICM0RG50</td><td></td></tr><tr><td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td><td>ThermoFisher scientific</td><td>168-0045</td><td></td></tr><tr><td>Nikon A1R confocal laser microscope system</td><td>Nikon</td><td></td><td></td></tr><tr><td>NIS-Elements Imaging Software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Acros Organics</td><td>41678-0010</td><td></td></tr><tr><td>Pasteur pipets</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>ThermoFisher scientific</td><td>P10144</td><td></td></tr><tr><td>Operating Scissors, straight</td><td>Harvard Apparatus</td><td>72-8403</td><td></td></tr><tr><td>Orbital shaker Belly Dancer</td><td>IBI Scientific</td><td>BDRLS0003</td><td></td></tr><tr><td>Prism 8</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Tissue Culture Dish, 60 mm w/ grip ring</td><td>Fisher Scientific</td><td>FB012921</td><td></td></tr><tr><td>Tissue culture plate, 24 well</td><td>Falcon/Corning</td><td>353047</td><td></td></tr><tr><td>Transfer pipettes, sterile</td><td>ThermoFisher scientific</td><td>13-711-21</td><td></td></tr><tr><td>Triton X-100</td><td>ThermoFisher scientific</td><td>BP151-500</td><td></td></tr></tbody></table>

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.

As shown in Figure 4, this protocol produces organotypic cerebellar slice cultures in which Purkinje cell survival can be assessed following the immunofluorescence and image acquisition steps. Purkinje cells were labeled with a combination of anti-Calbindin D-28K (dilution 1/200) and Alexa594 anti-mouse (dilution 1/300) antibodies. Image stitching was done automatically by the microscope acquisition software (NIS-Elements) to obtain a picture of the whole cerebellar slice. Purkinje cell numb...

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Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

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 ...

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Research

JoVE Journal

Neuroscience

7.9K Views.  Northwestern University, Feinberg School of Medicine. 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.

Video: Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Bioengineering

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Long-Term Engraftment of h-SC-NES Cells in Spinal Cord Organotypic Cultures

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs.
Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (&#8805;30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...

Cerebellar Purkinje Cells: DNA Damage Response in Organotypic Cultures

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

Author Spotlight: Deciphering the Role of ATM in Ataxia-Telangiectasia and the Associated Cerebellar Degeneration

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Summary

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Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

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