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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sterilization of materials
NOTE: All material and solutions must be sterilized prior to use.
<ol>
	<li>Sterilize all surgical tools and vibratome slicing material (knife holder, specimen disk, buffer tray) in a dry sterilizing oven for 4 h at 180 &#176;C.</li>
	<li>Sterilize temperature-sensitive material or equipment by UV or gamma irradiation.</li>
	<li>Sterilize media and solutions by autoclavation or filtration through 0.22 &#181;m pore membranes.</li>
</ol>
2. Preparation of solutions
<ol>
	<li>Prepare 15-20 mL of transport solution: 50% v/v Hanks&#39; balanced salt solution (HBSS) pH 7.4, 50% v/v basal medium for maintenance of post-natal and adult brain neurons (Table of Materials), supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3 mg/mL glucose, and 33 &#181;g/mL gentamicin. 
	NOTE: Transport solution must be refrigerated and oxygenated (bubbling with carbogen gas) for at least 20 min prior to sample collection.</li>
	<li>Prepare 300 mL of slicing solution (HBSS supplemented with 10 mM HEPES and 3 mg/mL glucose) and cool it down in a freezer to the point of initial crystal formation.</li>
	<li>Prepare 20 mL of culture medium: basal medium for maintenance of post-natal and adult brain neurons (Table of Materials) supplemented with 1% L-glutamine derivative (Table of Materials), 2% supplement for neural culture (Table of Materials), 1% penicillin/streptomycin, and 0.25 &#181;g/mL amphotericin B.</li>
</ol>
3. Setting up the slicing apparatus
NOTE: This protocol is ideally performed with the assistance of a colleague due to the logistics of sample collection in the surgical room.
<ol>
	<li>In a bucket of salt-added ice, let the slicing solution rest under carbogen mixture bubbling (95% O2, 5% CO2) for at least 20 min prior to use.</li>
	<li>Prepare a block of 3% agarose (approximately 2 cm x 2 cm x 2 cm) and superglue it to the vibratome specimen disk in order to create additional mechanical support to the tissue sample during slicing (Figure 1E).</li>
	<li>Set the vibratome for slicing: section thickness of 200 &#181;m, frequency of vibration of 100 Hz, and speed of slicing between 0.5-1.0 mm/s.</li>
	<li>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.</li>
</ol>
4. Sample collection
NOTE: In this protocol, human neocortical tissue was collected in the surgical room and transported to the laboratory.
CAUTION: When dealing with human samples, follow the appropriate safety protocols established by the Institution.
<ol>
	<li>Set up the transport apparatus (Figure 1C) that consists of: a portable gas cylinder with carbogen mixture connected to a pressure/flux valve that controls the gas output connected to a silicon tubing that connects gas output to the transport vessel; a transport vessel, usually a 50 mL conical centrifuge tube with perforated lid for gas input, containing the transport solution; and ice for sample cooling during transport.</li>
	<li>Collect and transport the specimen (Figure 1B) immediately to the lab. Submerse the specimen in cold transport solution (constantly bubbled with carbogen mixture).</li>
</ol>
5. Slicing
<ol>
	<li>Transfer the specimen to a Petri dish (100 mm x 20 mm) containing slicing solution and, with fine surgical tools, carefully remove as much as possible of the remaining meninges in the sample (Figure 1D).</li>
	<li>Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design, and with a no. 24 scalpel blade, trim a flat surface to be the base glued to the specimen disk.</li>
	<li>Using a disposable plastic spoon and delicate paintbrushes, collect the fragment from the Petri dish and dry excess solution using filter paper (dry by capillarity and avoid touching the tissue fragment with paper).</li>
	<li>Using superglue, attach the tissue to the vibratome specimen disk until it is firmly adhered to the disk and in contact with the agarose block (Figure 1E).</li>
	<li>Place the vibratome specimen disk (with tissue properly attached) in the vibratome buffer tray filled with slicing solution that must be bubbling during the whole process.</li>
	<li>Lock the knife holder in place with the razor blade firmly fixed.</li>
	<li>The slicing solution must cover both the specimen and the blade, only then start slicing (Figure 1F).</li>
	<li>Cut the specimen into 200 &#181;m slices. 
	NOTE: Although some vibratomes cut the specimens automatically, the close observation and minor adjustments in slicing speed during the process may help producing better slices. Discard initial irregular slices.</li>
	<li>Transfer the slices from the buffer tray to a Petri dish with slicing solution and trim loose edges and excess white mater to a proportion of around 70% cortex/30% white matter.</li>
</ol>
6. Culture
NOTE: Perform this step in a laminar flow cabinet under sterile environment.
<ol>
	<li>Add 600 &#181;L of culture medium per well (in a 24 well plate) and incubate for at least 20 min at 36 &#176;C and 5% CO2 prior to plating the slices.</li>
	<li>Plate one slice per well using a paintbrush (Figure 1G).</li>
	<li>If there are any unused wells in the plate, fill them with 400 &#181;L of sterile water.</li>
	<li>Incubate the plate at 36 &#176;C, 5% CO2. 
	NOTE: First medium replacement must be done in between 8-16 h after plating depending on the size of the slice.</li>
	<li>Supplement 10 mL of the previously prepared culture medium with 50 ng/mL brain derived neurotrophic factor (BDNF). 
	NOTE: During the first 8-16 h, the slices are incubated in 600 &#181;L of medium to avoid nutrient deprivation and acidification, since medium consumption in this phase is accelerated. From the next step, the volume of medium per well is adjusted to 400 &#181;L.</li>
	<li>Remove 333 &#181;L of the conditioned medium from each well and add 133 &#181;L of fresh BDNF-supplemented medium.</li>
	<li>Repeat the process of medium replacement every 24 h by replacing one-third of the conditioned medium with fresh BDNF-supplemented medium.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Sigma Aldrich</td>
			<td>SRP3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>108337</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salts</td>
			<td>Sigma Aldrich</td>
			<td>H1387-10X1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer pH 7.2</td>
			<td>Laborclin</td>
			<td>590338</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomicin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment and Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen Mixture</td>
			<td>White Martins</td>
			<td></td>
			<td>95% O2, 5% CO2</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>New Brunswick Scientific</td>
			<td>CO-24</td>
			<td>Incubation of slices 5% CO2, 36&#186;C</td>
		</tr>
		<tr>
			<td>Plastic spoon</td>
			<td></td>
			<td></td>
			<td>Size of a dessert spoon</td>
		</tr>
		<tr>
			<td>Razor Blade</td>
			<td>Bic</td>
			<td></td>
			<td>Chrome Platinum, used in slicing with vibratome</td>
		</tr>
		<tr>
			<td>Scalpel Blade</td>
			<td>Becton Dickinson (BD)</td>
			<td>Number 24</td>
			<td>Used for slicing of tissue; recommended same size or smaller</td>
		</tr>
		<tr>
			<td>Superglue (Loctite Super Bonder)</td>
			<td>Henkel</td>
			<td></td>
			<td>Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6&#39;-di-terc-butil-2,2&#39;-metilenodi-p-cresol; homopolymer</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>14047235612 - VT1000S</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparing free-floating slice cultures from an adult human brain

Source: Fernandes, A., et al. <a href="https://app.jove.com/v/59845">Short-Term Free-Floating Slice Cultures from the Adult Human Brain.</a> J. Vis. Exp. (2019).
This video demonstrates a technique for preparing free-floating slice cultures from the adult human brain. Thin slices are generated from a brain section using a vibratome. These slices are then suspended in a culture medium supplemented with growth factors to maintain tissue viability.

<img alt="Figure 1" src="/files/ftp_upload/22375/22375_Figure1.jpg" /> 
Figure 1: Sample collection, transport, slicing, and culturing of cortical tissue from adult humans. The procedure starts at the surgical room with collection of cortical tissue from temporal lobectomy for the treatment of pharmacoresistant epilepsy (A,B). (C) Tissue fragment (n = 1) is immediately transferred to a tube containing ice-cold oxygenated transport medium (see below). (D) In the lab, ...

Preparing Free-Floating Slice Cultures from an Adult Human Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sterilization of materials
NOTE: ...

Take a human brain tissue immersed in a slicing solution. The solution&#39;s biological pH and nutrients help maintain tissue functionality.
Remove the outermost meninges layer, and trim the tissue to create a flat surface.
Blot excess solution from the tissue. Attach the trimmed tissue to a specimen disk with an agarose block, keeping the tissue in contact with the block for ease of sectioning.
Place the disk in a vibratome buffer tray and cover the tissue with an ice-cold slicing solution.
Using the vibratome, generate uniform slices that preserve the tissue architecture.
Trim off excess tissue, ensuring a consistent proportion of gray and white matter.
Place the slices in a multiwell plate containing a culture medium and incubate.
The slices remain suspended in the medium to ensure even nutrient access, a technique known as free-floating slice culture.
Regularly replenish the culture with a neural growth factor-supplemented medium to maintain tissue viability.

preparing-free-floating-slice-cultures-from-an-adult-human-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

110 Views. Źródło: Fernandes, A., et al. Krótkoterminowe swobodnie pływające kultury warstwowe z mózgu dorosłego człowieka. J. Vis. Exp. (2019). Ten film demonstruje technikę przygotowywania swobodnie pływających kultur warstwowych z mózgu dorosłego człowieka. Cienkie plasterki są generowane z części mózgu za pomocą wibratomu. Wycinki te są następnie zawieszane w pożywce hodowlanej uzupełnionej czynnikami wzrostu w celu utrzymania żywotności tkanek. 

Video: Przygotowanie swobodnie unoszących się kultur warstwowych z dorosłego ludzkiego mózgu - Concept

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 ...

JoVE Journal

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

Medicine

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Preparing Free-Floating Slice Cultures from an Adult Human Brain

Transkrypcja

Preparing Free-Floating Slice Cultures from an Adult Human Brain