This work is supported by FAPESP (Grant 25681-3/2017 to AS), CAPES (Post-Doctoral fellowship PNPD/INCT-HSM to A.F. and Pre-Doctoral fellowship to N.D.M.) and FAEPA. G.M.A. holds a Master&#8217;s fellowship from FAPESP (MS 2018/06614-4). N.G.C. holds a CNPq Research Fellowship. We thank the patients and their families for donating the resected tissues for this study. We would like to acknowledge the support of residents, nurses, technicians, and the CIREP team, from the Clinical Hospital at the Ribeir&#227;o Preto Medical School, University of S&#227;o Paulo, who helped in various stages of the process.

Live adult brain tissues were obtained from patients undergoing resective neurosurgery for the treatment of pharmacoresistant temporal lobe epilepsy (Figure 1A). All procedures were approved by the Ethics Committee from the Clinics Hospital at the Ribeir&#227;o Preto Medical School (17578/2015), and patients (or their legal responsible person) agreed and signed the informed consent terms. Collection of the tissue was done by the neurosurgery team at the Epilepsy Surgery Cent...

The authors have nothing to disclose.

This protocol for producing free-floating, short-term slice cultures is an alternative method for culturing adult human neocortical slices. Such a protocol for slice cultures may be amenable for studies on (but not restricted to) optogenetics1,44,45, electrophysiology2,3,4,5, short-term plasticity<sup ...

The use of human samples in research is unequivocally a great option to study human brain pathologies, and modern techniques have opened new ways for robust and ethical experimentation using patient-derived tissue. Methods like organotypic/slice cultures prepared from adult human brain have been increasingly used in paradigms such as optogenetics1, electrophysiology2,3,4,5, plasticity6,7,8,9, neurotoxicity/neuroprotection10,11,12,13, cell therapy14, drug screening15,16,17, genetics and gene editing12,18,19,20, among others, as a strategy for better understanding neurological diseases during adulthood.
The comprehension of mechanisms underlying human brain pathologies depends on experimental strategies that require a large number of subjects. Conversely, in the case of slice cultures, although access to human samples is still difficult, the possibility of generating up to 50 slices from a single cortical sample partially circumvents the requirement of recruiting multiple volunteers by increasing the number of replicates and performed assays per collected tissue21.
Several protocols for brain organotypic/slice cultures have been described, ranging from the classical oculo drafts22,23 to roller tube24,25,26, semi-permeable membranes interface27,28,29,30, and free-floating slices31,32. Depending on the particularities of an experimental design, each technique has its own advantages and disadvantages. Short-term, free-floating slices cultures from adult human brains is in some cases advantageous over the method used by Stoppini et al.27, if considering the fact that although long-term cell survival in vitro is usually a major concern when evaluating a culture method, in many experiments only short periods of time in culture are needed12,31,32,33,34,35. Under these conditions, the use of free-floating cultures presents the advantage of being simpler and more cost-effective, as well as more accurately resembling the original human tissue condition than slices kept in culture over 2-3 weeks.
Despite the potential of slice cultures to neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly from human subjects. This article describes a protocol to use collected brain tissue from living human donors submitted to resective brain surgery to prepare free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are detailed. This protocol has been proven valuable for analyzing viability and neuronal function in investigations on the mechanisms of neuropathologies linked to adulthood.

<table border="0" cellpadding="0" cellspacing="0" style="width:1571px;" width="1570">
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		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">2-Propanol</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">1096341000</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Acrylamide/Bis-Acrylamide, 30% solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">A3449&#160;</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Ammonium persulfate</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">A3678-25G</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Amphotericin B</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:344px;">15290-018</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody anti-ERK 2 (rabbit)</td>
			<td>Santa Cruz Biotecnology</td>
			<td>sc-154</td>
			<td>Dilution 1:1,000 in BSA 2.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody anti-pERK (mouse)</td>
			<td>Santa Cruz Biotecnology</td>
			<td>sc-7383</td>
			<td>Dilution 1:1,000 in BSA 2.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BDNF</td>
			<td>Sigma Aldrich</td>
			<td>SRP3014</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Bovine Serum Albumin</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">A7906</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Bradford 1x Dye Reagent</td>
			<td style="width:200px;">BioRad</td>
			<td style="width:344px;">500-0205</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">EDTA</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">T3924</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose</td>
			<td>Merck</td>
			<td>108337</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#39;s Balanced Salts</td>
			<td>Sigma Aldrich</td>
			<td>H1387-10X1L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Hydrochloric acid</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">1003171000</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Hydrogen Peroxide (H2O2)</td>
			<td style="width:200px;">Vetec</td>
			<td align="right" style="width:344px;">194</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Mouse IgG, HRP-linked whole Ab (anti-mouse)</td>
			<td style="width:200px;">GE</td>
			<td style="width:344px;">NA931-1ML</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">NaCl</td>
			<td style="width:200px;">Merck</td>
			<td align="right" style="width:344px;">1064041000</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal A</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Non-fat dry milk (Molico)</td>
			<td style="width:200px;">Nestl&#233;</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Used for membrane blocking</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">PBS Buffer pH 7,2</td>
			<td>Laborclin</td>
			<td align="right">590338</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicilin/Streptomicin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:521px;">Potassium Chloride</td>
			<td>Merck</td>
			<td align="right">1049361000</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:521px;">Prime Western Blotting Detection Reagent</td>
			<td style="width:200px;">GE</td>
			<td style="width:344px;">RPN2232</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Rabbit IgG, HRP-linked whole Ab (anti-rabbit)</td>
			<td>GE</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:521px;">SDS</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">L5750</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:521px;">TEMED</td>
			<td style="width:200px;">GE</td>
			<td style="width:344px;">17-1312-01</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thiazolyl Blue Tetrazolium Bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>M5655</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Tris</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">T-1378</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:521px;">Triton x-100</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">X100</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:521px;">Ultrapure Water</td>
			<td style="width:200px;">Millipore</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Sterile water, derived from MiliQ water purification system</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:521px;">Equipment and Material</td>
			<td style="width:200px;"></td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;">24-well plates</td>
			<td>Corning</td>
			<td>CL S3526</td>
			<td>Flat Bottom with Lid</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Amersham Potran Premium (nitrocellulose membrane)&#160;</td>
			<td>GE</td>
			<td>29047575</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:521px;">Carbogen Mixture</td>
			<td style="width:200px;">White Martins</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">95% O2, 5% CO2</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;width:521px;">CO2 incubator</td>
			<td style="width:200px;">New Brunswick Scientific</td>
			<td style="width:344px;">CO-24</td>
			<td style="width:505px;">Incubation of slices 5% CO2, 36&#186;C</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:521px;">Microplate Reader</td>
			<td style="width:200px;">Molecular Devices</td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:521px;">Microtubes</td>
			<td style="width:200px;">Greiner</td>
			<td style="width:344px;">001608</td>
			<td style="width:505px;">1,5mL microtube</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:521px;">Motorized pestle</td>
			<td style="width:200px;">Kimble Chase</td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:521px;">Plastic spoon</td>
			<td style="width:200px;"></td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Size of a dessert spoon</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:521px;">Razor Blade</td>
			<td style="width:200px;">Bic</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Chrome Platinum, used in slicing with vibratome</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:37px;width:521px;">Scalpel Blade</td>
			<td style="width:200px;">Becton Dickinson (BD)</td>
			<td style="width:344px;">Number 24</td>
			<td style="width:505px;">Used for slicing of tissue; recommended same size or smaller</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:521px;">Superglue (Loctite Super Bonder)</td>
			<td style="width:200px;">Henkel</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6&#39;-di-terc-butil-2,2&#39;-metilenodi-p-cresol; homopolymer</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Vibratome&#160;</td>
			<td>Leica</td>
			<td>14047235612 - VT1000S</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:37px;width:521px;">Name of Material/ Equipment for Immunohistochemistry</td>
			<td style="width:200px;"></td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;">Antibody anti-NeuN (mouse)</td>
			<td>Millipore&#160;</td>
			<td>MAB377</td>
			<td>Dilution 1:1,000 in Phosphate Buffer</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:521px;">Antibody anti-GFAP (mouse)</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">MAB360</td>
			<td>Dilution 1:1,000 in Phosphate Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Antibody anti-Iba1 (rabbit)</td>
			<td style="width:200px;">Abcam</td>
			<td style="width:344px;">EPR16588 - ab178846</td>
			<td>Dilution 1:2,000 in Phosphate Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Biotinylated anti-mouse IgG Antibody (H+L)</td>
			<td style="width:200px;">Vector</td>
			<td style="width:344px;">BA-9200</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">DAB</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">D-9015</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Entellan</td>
			<td style="width:200px;">Merck</td>
			<td align="right" style="width:344px;">107960</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Ethanol</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">1.00983.1000</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Gelatin</td>
			<td style="width:200px;">Synth</td>
			<td style="width:344px;">00G1002.02.AE</td>
			<td style="width:505px;">Used for coating slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome</td>
			<td>Leica</td>
			<td>SM2010R</td>
			<td>Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum</td>
			<td>Jackson Immuno Research</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Paraformaldehyde</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">158127</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Rabbit IgG, HRP-linked whole Ab (anti-rabbit)</td>
			<td>GE</td>
			<td>NA934-1ML</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slides (Star Frost)</td>
			<td>Knittel Glaser</td>
			<td></td>
			<td>Gelatin coated slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td style="width:200px;">Vetec</td>
			<td>60REAVET017050</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Vectastain ABC HRP Kit (Peroxidase, Standard)</td>
			<td style="width:200px;">Vector</td>
			<td style="width:344px;">PK-4000, Kit Standard</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Xylene</td>
			<td style="width:200px;">Synth</td>
			<td style="width:344px;">01X1001.01.BJ</td>
			<td style="width:505px;"></td>
		</tr>
	</tbody>
</table>

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 critical aspect to evaluate the quality and health of cultured slices is the presence and typical morphology of the expected neural cell types, neurons, and glial cells. The typical architecture of the human cortical lamination was observed in a slice at DIV4, revealed by neuronal immunolabeling (Figure 2D). In addition, the expected presence of microglia and astroglia (Figure 2B,C) was also observed. These results demonstrate...

Watch this Scientific Journal Video about Short-Term Free-Floating Slice Cultures from the Adult Human Brain at JoVE.com

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

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 ...

We present a protocol to prepare free-floating slice cultures from brain tissue collected from living human donors submitted to resective brain surgery. This culture is amenable to perform biochemical and cell biology assays or immunohistochemistry. It is expected to contribute to the elucidation of the mechanism underlying neurodegeneration in associated brain diseases.The main advantage of this technique is that it's a simpler and cost effective alternative to the classic slice culture protocol using membrane inserts. Although the overall protocol is not complex, some steps such as removal of meninges, gluing the tissue to the vibratome specimen disc, and resection for immunohistochemistry are best understood when demonstrated visually. Helping to demonstrate the procedure will be Niele Mendes and Glaucia Almedia who are grad students, and Giovanna Nogueira, another grad student.To begin this procedure, add salt to a bucket of ice. Transfer the slicing solution to this bucket and let it rest under carbogen mixture bubbling for at least 20 minutes prior to use. Next, prepare a block of 3%agarose that is about two centimeters by two centimeters by two centimeters.Super glue the agarose block to the vibratome specimen disc in order to create additional mechanical support to the tissue sample during slicing. On the vibratome, set the section thickness to 200 micrometers, the vibration frequency to 100 hertz, and choose a slicing speed between 0.5 and 1.0 millimeters per second. Then lock the vibratome buffer tray to the vibratome base and add ice to keep it refrigerated prior to receiving the slicing solution and the sample and throughout the slicing procedure.First, set up a transport apparatus that consists of a portable gas cylinder containing a carbogen mixture connected to a pressure flux valve that controls the gas output connected to silicone tubing that connects that gas output to the transport vessel and the transport vessel which contains the transport solution and ice for sample cooling during transport. Collect and transport the specimen and transport as outlined in the text protocol. Transfer the specimen to a Petri dish containing slicing solution.Using fine surgical tools, carefully remove as much of the remaining meninges as possible. Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design and use a number 24 scalpel blade to trim a flat surface to be the base that will be glued to the specimen disc. Using a disposal plastic spoon and delicate paintbrushes, collect the fragment from the Petri dish and dry off excess solution with filter paper.Next, use super glue to attach the tissue to the vibratome specimen dish until it is firmly adhered to the dish and in contact with the agarose block. Place the vibratome specimen disc into the vibratome buffer tray. Lock the knife holder in place with the razor blade firmly fixed.Add slicing solution and ensure that it is covering both the specimen and the blade. Then begin slicing the specimen into 200 micrometer slices. Transfer the slices from the buffer tray to a Petri dish containing slicing solution and trim any loose edges and excess white matter to a proportion of approximately 70%cortex and 30%white matter.In a laminar flow cabinet, add 600 microliters of culture medium to each well of a 24-well plate and incubate at 36 degrees Celsius with 5%carbon dioxide for at least 20 minutes prior to plating the slices. After this, use a paintbrush to plate one slice in each well. If there are any unused wells in the plate, fill them with 400 microliters of sterile water.Incubate at 36 degrees Celsius with 5%carbon dioxide. Supplement 10 milliliters of the previously prepared culture medium with brain derived neurotrophic factor at a concentration of 50 nanograms per milliliter. After eight to 16 hours, remove 333 microliters of the conditioned medium from each well and add 133 microliters of fresh BDNF supplemented medium.Replace one-third of the conditioned medium with fresh BDNF supplemented medium every 24 hours. First, transfer the slices from the wells containing culture medium to a new 24-well plate containing PBS. Remove the PBS from each well and replace it with one milliliter of 4%paraformaldehyde.Incubate overnight at four degrees Celsius. The next day, carefully remove the paraformaldehyde from the wells and replace it with one milliliter of 30%sucrose solution. Incubate at four degrees Celsius for 48 hours.After 48 hours, set the freezing microtome to negative 40 degrees Celsius. Prepare a sucrose base on the microtome stage where the slices should be placed. After it is frozen completely, cut some of the frozen sucrose to produce a flat surface on which the slice will be placed.Next, place each slice over a stretched plastic film and use a paintbrush to flatten the tissue. In a single move, transfer the stretched slice to the frozen sucrose base. After the slice has rested for five to 10 minutes to freeze properly, cut the slice into 30 micrometer sections.Transfer the 30 micrometer sections to a Petri dish containing PBS and proceed to a histology protocol adequate for the experimental design. When determining the quality and health of cultured slices, a critical aspect to evaluate is the presence and typical morphology of the expected neural cell types, neurons and glial cells. The typical architecture of the human cortical lamination is observed in a slice at day in vitro four revealed by neuronal immunolabeling.In addition, the expected presence of microglia and astroglia is also observed. These results demonstrate that tissue architecture is not significantly affected either by the surgical procedure, the sample processing, or by the short-term period in vitro. The neuronal response to potassium chloride-induced depolarization have also been quantified in cultured slices by following ERK phosphorylation.Interestingly, a clear increase in ERK phosphorylation is seen in potassium chloride-treated slices at day in vitro four. Finally, the response of slices at day in vitro four to a toxic challenge is evaluated with a known oxidative stress inducer hydrogen peroxide. Exposing the slices to 300 millimolar hydrogen peroxide for 24 hours led to a robust decrease in MTT reduction.Taken together, the massive cell death observed in day in vitro five slices after the hydrogen peroxide challenge and the potassium chloride-induced depolarization results indicate the preserved general health of slices at day in vitro four which respond to a toxic stimulus such as oxidative stress. This protocol is mainly devoted to studies based on assays lasting than one week such as the investigation of molecular mechanisms of neurotoxicity and neuroprotection by candidate drugs. Although this protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment for micro resistant temporal lobe epilepsy, it's likely that tissue collected from other brain regions or conditions could also be sources to produce free-floating slice cultures.Slice cultures from adult human brain may be key in advancing our understanding of human neuropathologies due to their unique cellular circuitry and molecular machinery lacking in slices produced from rodent brains.

short-term-free-floating-slice-cultures-from-the-adult-human-brain

Research

JoVE Journal

Neuroscience

8.6K ビュー.  University of São Paulo. 我々は、切除脳外科手術に提出された生きているヒトドナーから採取された脳組織から自由に浮遊スライス培養を準備するためのプロトコルを提示する。この培養は、生化学的および細胞生物学のアッセイまたは免疫細胞化学を行うのもよい。関連する脳疾患における神経変性の根底にあるメカニズムの解明に寄与することが期待される。この技術の主な利点は、?...