Name: short-term free-floating slice cultures from the adult human brain
Uploaded: 2019-11-05T13:30:00
Description: Watch this Scientific Journal Video about Short-Term Free-Floating Slice Cultures from the Adult Human Brain at JoVE.com

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...

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.

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			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">T3924</td>
			<td style="width:505px;">Used in RIPA buffer</td>
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			<td height="40" style="height:40px;">Glucose</td>
			<td>Merck</td>
			<td>108337</td>
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			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
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			<td>Sigma Aldrich</td>
			<td>H1387-10X1L</td>
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			<td height="40" style="height:40px;">Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Hydrochloric acid</td>
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			<td style="width:344px;">1003171000</td>
			<td style="width:505px;"></td>
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		<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>
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			<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>
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			<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>
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			<td height="40" style="height:40px;">Neurobasal A</td>
			<td>Gibco</td>
			<td>10888-022</td>
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		<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>
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			<td height="40" style="height:40px;width:521px;">PBS Buffer pH 7,2</td>
			<td>Laborclin</td>
			<td align="right">590338</td>
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			<td height="40" style="height:40px;">Penicilin/Streptomicin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
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			<td height="32" style="height:32px;width:521px;">Potassium Chloride</td>
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			<td align="right">1049361000</td>
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			<td height="31" style="height:31px;width:521px;">Prime Western Blotting Detection Reagent</td>
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			<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>
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		<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>
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			<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>
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			<td height="40" style="height:40px;">Thiazolyl Blue Tetrazolium Bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>M5655</td>
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			<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>
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			<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>
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		<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>
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		<tr height="38">
			<td height="38" style="height:39px;width:521px;">Equipment and Material</td>
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			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
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			<td height="49" style="height:50px;">24-well plates</td>
			<td>Corning</td>
			<td>CL S3526</td>
			<td>Flat Bottom with Lid</td>
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			<td height="44" style="height:44px;">Amersham Potran Premium (nitrocellulose membrane)&#160;</td>
			<td>GE</td>
			<td>29047575</td>
			<td></td>
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		<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>
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		<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>
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			<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>
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			<td style="width:505px;">Size of a dessert spoon</td>
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			<td height="39" style="height:39px;width:521px;">Razor Blade</td>
			<td style="width:200px;">Bic</td>
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			<td style="width:505px;">Chrome Platinum, used in slicing with vibratome</td>
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		<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>
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			<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>
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			<td height="39" style="height:39px;">Vibratome&#160;</td>
			<td>Leica</td>
			<td>14047235612 - VT1000S</td>
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		<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>
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		<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>
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		<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>
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		<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 ...

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Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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 ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...