Name: Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells
Uploaded: 2024-07-31T15:22:17
Description: The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use ...

establishing a three-dimensional culture of rat brain-derived glial cells

Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

24 hours before cell encapsulation, aspirate the medium from two-week primary cultures, and add 1 milliliter of fresh medium to each well. On the day of cell encapsulation, prepare 12.5 milliliters of dilute trypsin and 25 milliliters of medium for each 12-well plate, and warm to 37 degrees Celsius in a water bath. Then, use a 10-milliliter serological pipette to add around 1 milliliter of diluted trypsin to each well of the 12-well plate.
Incubate for 20 to 30 minutes at 37 degrees Celsius and 5% carbon dioxide until the confluent cell layer detaches from the plate. After the incubation, use a 1-milliliter pipette to recover the detached cells, which should appear as a single floating piece, and transfer to a 15-milliliter conical centrifuge tube. Dilute with an equivalent volume of medium and centrifuge at 200 times g for 2 minutes at room temperature.
Following centrifugation, discard the supernatant and resuspend the cell pellet in 10 milliliters of warm medium. Triturate the cells five times with the pipette, then, centrifuge again at 200 times g for two minutes. After the spin, decant the supernatant, resuspend the cells in 5 milliliters of warm medium, and transfer the cells to a 50-milliliter conical centrifuge tube.
Then, using a 10-milliliter syringe with an 18-gauge needle, triturate the cell solution three times and filter the suspension through a 40-micron cell sieve into a new conical centrifuge tube. Next, collect 10 microliters of cell suspension, and dilute 1-to-100 in warm medium, and count the cells with an automated cell counter.
Incubate the remaining cells in a 37 degrees Celsius water bath until the encapsulation step. Warm 12.5 milliliters of naive medium and 12.5 milliliters of conditioned medium for each plant 12-well plate of 3D hydrogels. Then, weigh 7 milligrams of HAMA per plant 12-well plate, and add to sterile filtered for a concentration of 2% weight for volume. Sonicate the solution for 60 minutes at 20 kilohertz until the HAMA is fully dissolved.
While sonicating, prepare individual 10% solutions of both TEA and NVP. Also prepare a 1-millimolar solution of eosin in 1-milliliter aliquots of sterile PBS. Then, for each 12-well plate, make a final 1.4 1.4-milliliter mixture of 1 x 107 cells, 0.5% HAMA, 20% basal lamina mixture in PBS, 0.1% TEA, 0.1% NVP, and 0.01 millimolar eosin Y.
After gently mixing the solution, use a pipette fitted with a 1-milliliter tip to dispense 100 microliters into each PDMS mold. Then, expose the samples to a high-intensity green LED light in an enclosed box for 5 minutes at room temperature.
The five-minute green LED exposure is essential to form the 3D gel. The red color introduced by the eosin Y will become transparent and indicate the termination of the reaction.
Next, add 1 milliliter of warm, conditioned media to each well of a 12-well plate; grasp each coverslip between thumb and forefinger and use curved tweezers to slowly peel off the PDMS mold without displacing the gel. Place the mold into a well with conditioned medium.
Removing the mold is the most challenging step. It should be done with utmost care to prevent dislodging the gel and fracturing the coverslip. This may take practice.
After all molds have been transferred to the wells of the plate, add an additional 1 milliliter of warm DMEM/F12 medium to each well. Incubate the plates at 37 degrees Celsius with 5% carbon dioxide for two weeks before imaging.

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The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use Committee at the University of Alberta.
1. Microglia and Astrocyte Isolations
NOTE: All media for isolation and cell culture is preheated to 37 &#176;C in a water bath. Hank&#39;s balanced salt solution (HBSS) has 1% penicillin-streptomycin (PS). All Dulbecco&#39;s modified Eagle&#39;s media with Ham&#39;s F12 nutrient mixture (DMEM/F12) is supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). Only mixtures with trypsin lack FBS. All materials in this protocol are sterile (filter, alcohol, purchased, and autoclaved) and every step subsequent to step number 1.8 is performed in a biosafety cabinet with aseptic technique.
<ol>
	<li>Per brain, pre-heat 15 mL of HBSS and 12.5 mL DMEM/F12 to 37 &#176;C in 50 mL conical centrifuge tubes, and approximately 2 mL of 0.25% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA) in a 15 mL conical centrifuge tube. Heat an additional 25 mL of DMEM/F12. Warm trypsin about 10 min prior to use to prevent loss of enzymatic activity.</li>
	<li>Pour enough warm HBSS into sterile, 6 and 10 cm culture dishes to cover the surface. Prepare one 10 cm dish for every two brains plus an additional 6 cm dish.</li>
	<li>Place up to 2 brains in each 10 cm2&#160;dish (Figure 1A).</li>
	<li>Under a dissection microscope, use curved and straight forceps to separate the cortices along the midline, and the cerebellum with brain stem. This yields three sections of tissue per brain (Figure 1B).</li>
	<li>Peel the thin, semi-transparent, layer (meninges) of tissue containing blood vessels from each section of tissue and discard, transferring the remaining tissue into a separate culture dish. Grasp the thin layers exposed by the tissue cuts, with the forceps, and carefully pull until major portions are &#39;hanging off&#39; the brain tissue. Finally remove this portion by gently tugging. See&#160;Figure 1C-1E&#160;for representative images of before, during, and after this step.</li>
	<li>In a biosafety cabinet, remove the remaining HBSS from the culture dish, carefully retaining the tissue. Macerate the tissue with a scalpel and transfer tissue to the pre-warmed 15 mL tube with trypsin.</li>
	<li>Incubate this tube for 25 min in a 37 &#176;C bath to digest the tissue enzymatically.</li>
	<li>Centrifuge the tube down at 500 x g for 2 min at room temperature, and pour out the supernatant.</li>
	<li>Using a 10 mL serological pipette, add 10 mL of warm DMEM/F12 to inactivate the trypsin and triturate the solution 5 times to break up the tissue.</li>
	<li>Centrifuge the tube down at 500 x g for 2 min at room temperature, and pour out the supernatant.</li>
	<li>Using a 10 mL serological pipette, re-suspend pellet into 10 mL of warm DMEM/F12 and transfer the solution to a 50 mL Conical centrifuge tube.</li>
	<li>Using a 10 mL syringe with an 18 gauge needle, triturate the solution 3 times.</li>
	<li>Distribute this solution into increments of 12.5 mL of warm DMEM/F12 per brain.</li>
	<li>Pipette 1 mL increments of the triturated solution into a 12 well plate (1 per brain).</li>
	<li>Incubate at 37 &#176;C and 5% CO2 in a humidified cell culture incubator. Replace media after three days, and refresh media every subsequent three days. After 2 weeks, the cells can be collected for experiments.</li>
</ol>
2. Macromer Synthesis
<ol>
	<li>Weigh 375 mg of hyaluronic acid (HA) salt in a 50 mL conical centrifuge tube and add 37.5 mL of distilled water.</li>
	<li>Flush the solution with a vortexer for 1 min and sonicate the mixture until it appears homogenous and loses its gel-like viscosity. This process may take 6 h.</li>
	<li>Transfer solution to a 100 mL beaker with a magnetic stir bar (38 mm), and stir vigorously at room temperature.</li>
	<li>Carefully titrate 5.0 M NaOH into the mixing HA and measure with pH paper until the pH stabilizes between 8 and 12.</li>
	<li>Collect 3 mL of fresh methacrylic anhydride (MA, 94%), blanketed by nitrogen during storage. 
	NOTE: MA is exposed to the atmosphere for long durations (days), it will lose its reactivity. 
	CAUTION: All use of MA should be done in a fume hood.</li>
	<li>Add 200 &#181;L of MA into the mixing HA solution. The reaction lowers the pH of the solution below 8.</li>
	<li>Repeat steps 2.4 to 2.6 until the 3 mL of MA have been added to the solution. This process may take up to 1-2 h.</li>
	<li>Allow the reaction to continue overnight at 4 &#176;C, without mixing.</li>
	<li>Transfer solution to 400 mL of cold ethanol (EtOH) in a 500 mL beaker and allow precipitation for 24 - 72 h at 4 &#176;C.</li>
	<li>Carefully decant the major portion of the solution into a separate beaker for disposal, and collect the precipitate in a 50 mL conical centrifuge tube. This may be distributed in 2-4 tubes, if necessary.</li>
	<li>Centrifuge the tubes at 200 x g in a centrifuge for 2 min at room temperature and dispose of the supernatant.</li>
	<li>Top up the tube with cold EtOH, mix the solution with a vortexer for 1 min, and repeat step 2.10-2.11.</li>
	<li>Add 25 mL of sterile deionized water, mix the solution with a vortexer for 1 min, sonicate (&#62;20 kHz) for 1 h, and incubate at 4 &#176;C overnight.</li>
	<li>Place the solution in a -80 &#176;C freezer for 15 min or until frozen or flash freeze in liquid nitrogen. 
	CAUTION: When using liquid nitrogen, use protective gloves, a face shield, and an apron.</li>
	<li>Freeze dry the tube (below 0.1 mBar and -30 &#176;C) for 24 - 48 h until a snow-like powder is produced.</li>
	<li>At this point, test the sample for purity via&#160;1H nuclear magnetic resonance, where the amount of methacrylate protons (peaks at 6.1 and 5.6 ppm), relative to methyl protons (1.9 ppm), on the HA backbone can be confirmed. A minimum ratio of 95% methacrylate to methyl protons is acceptable.</li>
</ol>
3. Gel Formation and 3D Encapsulation
<ol>
	<li>Coverslip and Mold Preparation
	<ol>
		<li>Make a 50 mL distilled water solution of 2% 3-(Trimethoxysilyl) propyl methacrylate in a 50 mL conical centrifuge tube.</li>
		<li>Place 18 mm glass coverslips in solution and rock for 1 h at room temperature. Up to 50 coverslips can be added at once.</li>
		<li>Rinse coverslips by serially dipping each in 3 beakers of 100 mL deionized water.</li>
		<li>Dry the coverslips in a vacuum at 40 &#176;C overnight with a desiccator and oven.</li>
		<li>Quickly immerse individual coverslips in 70% EtOH with forceps.</li>
		<li>Without drying, drop each coverslip into a well of a 12 well plate.</li>
		<li>Wash and aspirate each well with sterile deionized water (1 mL per well).</li>
		<li>Add 1 mL of sterile 2 &#181;g /mL poly-L-lysine (PLL) to each well and incubate for &#62;2 h.</li>
		<li>Aspirate PLL solution and allow the coverslips to air dry.</li>
		<li>Immerse polydimethyl siloxane (PDMS) molds (see&#160;Figure 2) in 70% EtOH, and place one upon the center of each coverslip, and allow to air dry and create a seal between the mold and glass.
		<ol>
			<li>Prepare PDMS molds by casting PDMS premix reagents in a 10:1 ratio in a flat-bottomed polystyrene dish. The quantity of PDMS prepared is selected to yield a sheet approximately 1 mm thick. To form wells in the PDMS, cut the sheets with a circle punch with an inner diameter of 10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell Preparation 
	&#8203;NOTE: All steps in the protocol are performed with sterile technique in a biosafety cabinet. The phosphate buffered saline (PBS) solution is adjusted to pH 7.4 for all steps.
	<ol>
		<li>24 h prior to cell encapsulation in methacrylated hyaluronic acid (HAMA), refresh the medium for the 2-week primary cultures (from Step 1.15) in the 12 well plates. Add 1 mL of DMEM/F12 (10% FBS and 1% PS) medium per well.</li>
		<li>For each 12-well plate of cells prepare 12.5 mL of dilute trypsin (0.25% trypsin-EDTA diluted 30% with DMEM/F12 media), and 25 mL DMEM/F12 (10% FBS and 1% PS) and warm in at 37 &#176;C in a water bath.</li>
		<li>Collect conditioned medium from plates using a 10 mL serological pipette (&#62; 0.5 mL per well), thoroughly aspirate remaining liquid, and replace with 1 mL per well of dilute trypsin (0.075% trypsin-ethylenediaminetetraacetic acid [EDTA]) for 20-30 min in an incubator (37 &#176;C, 5% CO2) until the confluent cell layer detaches from the plate.</li>
		<li>Recover suspended cell material with a 1 mL pipette (appears as a single floating piece) and collect in a 15 mL conical centrifuge tube. Dilute with an equivalent volume of DMEM/F12 (10% FBS and 1% PS), and centrifuge at 200 x g for 2 min at room temperature, discarding the supernatant.</li>
		<li>Resuspend the cell pellet in 10 mL warm DMEM/F12 (10% FBS and 1% PS), triturate the cells 5 times with a 10 mL pipette, and centrifuge at 200 x g for 2 min.</li>
		<li>Decant the supernatant, re-suspend the cells in 5 mL warm DMEM/F12, and transfer the cells to a 50 mL conical centrifuge tube.</li>
		<li>Using a 10 mL syringe with a 18 gauge needle, triturate the cell solution 3 times, and filter the suspension through a 40 &#181;m cell sieve into a new conical centrifuge tube.</li>
		<li>Collect 10 &#181;L of cell suspension, dilute 1:100 in warm DMEM/F12, and count cells with an automated cell counter.</li>
		<li>Incubate in a 37 &#176;C water bath until encapsulation step.</li>
	</ol>
	</li>
	<li>Cell Encapsulation 
	&#8203;NOTE: All steps in the protocol are performed with sterile technique in a biosafety cabinet.
	<ol>
		<li>For each 12-well plate of 3D hydrogels warm 12.5 mL of DMEM/F12 and 12.5 mL of the conditioned DMEM/F12 media collected during cell preparation.</li>
		<li>Weigh the quantity of HAMA required for a final concentration of 0.5% wt/vol. Dissolve this HAMA in sterile filtered PBS at a concentration of (2% wt/vol); a concentration 4 times higher than the final concentration is utilized to accommodate the addition of cells and other reagents. One 12 well plate of hydrogels requires ~350 &#181;L PBS with 7 mg HAMA.</li>
		<li>Sonicate (&#62;20 kHz) solutions until HAMA is fully dissolved for 60 min.</li>
		<li>Prepare individual 10% solutions of both triethanolamine (TEA) and 1-vinyl-2-pyrrolidinone (NVP), and a 1 mM solution of Eosin Y (EY) in 1 mL aliquots of sterile PBS pH 7.4.</li>
		<li>For one 12 well plate, make a final 1.4 mL mixture of 1 x 107&#160;cells, 0.5% wt/vol HAMA (350 &#181;L of 2% wt/vol), 0.1% TEA (14 &#181;L of 10%), 0.1% NVP (14 &#181;L of 10%), and 0.01 mM EY (14 &#181;L of 1 mM), and 20% basal lamina mixture (280 &#181;L of stock). The remaining volume is PBS.</li>
		<li>Gently mix the solution and pipette 100 &#181;L into each PDMS mold with a 1 mL tip.</li>
		<li>Expose samples to a high intensity green LED light (~520 nm, 60 mW, in an enclosed 20 cm x 20 cm x 20 cm box) for 5 min at room temperature.</li>
		<li>Add 1 mL per well of warm conditioned DMEM/F12 to a 12 well plate.</li>
		<li>Grasp each coverslip between thumb and forefinger, slowly peel the PDMS mold off with curved tweezers being careful not to displace the gel and place it into a well with conditioned medium.</li>
		<li>Add an additional 1 mL warm DMEM/F12 to each well.</li>
		<li>Incubate plates at 37 &#176;C with 5% CO2&#160;for 2 weeks, refreshing cell media every week by pipetting 1 mL of media from each well and replacing with 1 mL DMEM/F12.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Materials for HAMA synthesis and photopolymerization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronic acid (HA)</td>
			<td>Sigma-Aldrich</td>
			<td>53747-10G</td>
			<td>Streptococcus equi, MW: 1.5 - 1.8 X 10^6</td>
		</tr>
		<tr>
			<td>Methacrylic anhydride (MA)</td>
			<td>Sigma-Aldrich</td>
			<td>275585-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>221465-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Commerical Alcohols Inc.</td>
			<td></td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (pH 7.4) tablets</td>
			<td>Fisher Scientific</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>90279-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Vinyl-2pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>EosinY (EY)</td>
			<td>Sigma-Aldrich</td>
			<td>E6003-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-(Trimethoxysilyl)propyl methacrylate</td>
			<td>Sigma-Aldrich</td>
			<td>440159-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (100 mL)</td>
			<td>Corning</td>
			<td>1000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (500 mL)</td>
			<td>Corning</td>
			<td>1000-600</td>
			<td></td>
		</tr>
		<tr>
			<td>pH paper (Labstick)</td>
			<td>Sigma-Aldrich</td>
			<td>9580</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Materials for glial cell isolation and cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P1-2 Sprague Dawley rat pups</td>
			<td>Charles River</td>
			<td>CD Sprague Dawley rat strain code 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissector scissors - slim blades (small)</td>
			<td>Fine Science Tools</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors - Toughcut (large)</td>
			<td>Fine Science Tools</td>
			<td>14130-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps (Dumont #5)</td>
			<td>Fine Science Tools</td>
			<td>11521-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved fine forceps (Dumont #7)</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium and Ham&#39;s nutrient mixture F-12 (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (PS)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (PLL)</td>
			<td>Sigma-Aldrich</td>
			<td>P-6282</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well Tissue culture treated plates (Cellstar)</td>
			<td>Greiner Bio-One</td>
			<td>665 108</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-676-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipette</td>
			<td>Fisher Scientific</td>
			<td>12-676-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (60 mm X 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (100 mm X 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslip (18 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-100 18CIR</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Koss, K. M, et al. <a href="https://app.jove.com/v/56615">Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to establish a three-dimensional culture of glial cells. Rat-brain-derived glial cells are encapsulated within a photocrosslinkable polymer matrix containing extracellular matrix components. Upon incubating in a growth medium, the glial cells proliferate within the supporting matrix to create a three-dimensional culture.

<img alt="Figure 1" src="/files/ftp_upload/22389/22389_Figure1.jpg" /> 
Figure 1: Dissection of day 1 Sprague Dawley rat pup brain.&#160;Images of whole brain (A), dissection of cortices and cerebellum (B), a single cortex with meninges (C), peeling of the meninges with forceps (D), and the meninge-free cortex (E).
<img alt...

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use ...

Take a microplate with an adherent culture of glial cells derived from a rat brain. The culture contains astrocytes, microglia, and oligodendrocytes.
Remove the medium, incubate with an enzyme solution to detach the cells, and then transfer them into a tube.
Centrifuge and remove the supernatant with cellular debris. Resuspend the cells in a warm medium.
Triturate using a syringe to break up cell clumps and filter using a strainer to obtain a single-cell suspension.
Add a photocrosslinkable biopolymer, crosslinking agents, and extracellular matrix components.
Pour the mixture into a mould attached to a coverslip. Expose the mixture to high-intensity light to induce crosslinking, forming a gel and encapsulating the glial cells.
Remove the mould and place the coverslips in a microplate. Add a warm medium and incubate the gel under physiological conditions.
The cells proliferate within the supporting extracellular matrix and create a cellular network, establishing a three-dimensional culture of glial cells.

24 Views. Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

Video: Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells - Experiment

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

JoVE Journal

Environment

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and tunable 3D hydrogel, suitable for culturing cells and tissue in a setting that matches their native environment. This is particularly important for researchers investigating the initiation, growth, and treatment of cancer where the interaction between cells and their local extracellular matrix is a fundamental part of the model. Moving to 3D culture can be challenging and is often associated with a lack of reproducibility due to high batch-to-batch variation in animal-derived 3D culture matrices. Similarly, handling issues can limit the usefulness of synthetic hydrogels. In response to this need, we have optimized a simple self-assembling peptide gel, to enable the culture of relevant cell line models of cancer and disease, as well as patient-derived tissue/cells. The gel itself is free from matrix components, apart from those added during encapsulation or deposited into the gel by the encapsulated cells. The mechanical properties of the hydrogels can also be altered independent of matrix addition. It, therefore, acts as a &#8216;blank slate&#8217; allowing researchers to build a 3D culture environment that reflects the target tissue of interest and to dissect the influences of mechanical forces and/or biochemical control of cell behavior independently.

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and ...

Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

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