an in vitro method to generate astrocyte scaffolds within hydrogel micro-columns

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

Inside a biosafety cabinet, prepare a 1 milligram per milliliter solution of rat tail type I collagen in co-culture medium and then place it on ice. Adjust the pH of the ECM solution with acid or base as necessary, until the pH is stable in the 7.2 to 7.4 range. Transfer the microcolumn boat to a 35 or 60-milliliter Petri dish with forceps. Using the stereoscope for guidance, situate the 10-microliter tip of a micropipette at one end of each microcolumn and suction to empty the lumen of DPBS and air bubbles.
Charge a P10 micropipette with the collagen solution. Place the 10-microliter tip against one end of each microcolumn, and deliver enough solution to fill the lumen, then pipette a reservoir of ECM on either end of the microcolumn. Once all columns have been filled, pipette co-culture medium in a ring around the Petri dish to prevent the columns from drying out.
Incubate the dish containing the microcolumns at 37 degrees Celsius and 5% carbon dioxide for at least one hour to promote polymerization of collagen before adding cells. Afterwards, place the tip of a P10 micropipette at one end of a microcolumn, and transfer approximately 5 microliters of cell solution into the lumen to fill it. After seeding all the microcolumns in a boat, incubate it at 37 degrees Celsius and 5% carbon dioxide for one hour to promote the attachment of astrocytes to the ECM.
After the incubation period, carefully fill the dishes containing the seeded microcolumns with 3 or 6 milliliters of co-culture media for 35 or 60-millimeter Petri dishes, respectively. Maintain the plated microcolumns in culture at 37 degrees Celsius and 5% carbon dioxide to promote the self-assembly of the aligned astrocytic bundles, which should form a bundled cable-like structure after 6 to 10 hours.

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1. Development of the Agarose Hydrogel Micro-columns
<ol>
	<li>Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a sterile beaker containing 100 mL of Dulbecco&#39;s phosphate buffered saline (DPBS). Add a sterile magnetic bar to the beaker, and place it on the surface of a hot plate/stirrer. Keep the beaker covered to prevent evaporation of its contents in the next step.</li>
	<li>Heat the agarose and DPBS at a temperature of 100 &#176;C and stir at 60-120 rpm. Adjust these settings as necessary, and constantly monitor the progression of the dissolution process as the solution changes initially from opaque to a clear appearance that signifies that the agarose has been completely dissolved. 
	Caution: The hot beaker and the solution are hot!</li>
	<li>As the agarose solution is heated and stirred, retrieve four empty 10 cm Petri dishes and add 20 mL of DPBS to two of them. Place acupuncture needles (diameter: 300 &#181;m, length: 40 mm), glass microliter capillary tubes (diameter: 701.04 &#181;m, length: 65 mm, capacity: 25.0 &#181;L), and a bulb dispenser inside the biosafety cabinet. When the liquid agarose solution clears out, maintain constant heating at approximately 50 &#176;C and stirring to avoid gelation of the agarose.</li>
	<li>Introduce an acupuncture needle into the bottom opening of a bulb dispenser. Insert a capillary tube over the needle exposed to the outside. Secure the capillary tube by entering part of it into the rubber section of the bulb dispenser cylinder.</li>
	<li>Transfer 1 mL of liquid agarose with a micropipette to the surface of an empty Petri dish, and place one end of the capillary tube vertically (with the needle inserted) in contact with agarose, while the rubber cap of the bulb dispenser is being pinched inwards. Slowly release the pressure on the bulb dispenser cap to draw agarose into the capillary tube. 
	NOTE: The transfer of liquid agarose into the capillary tubes must be performed rapidly. If the liquid agarose is left to cool on the Petri dish surface for sufficient time (approximately 60 s), it starts to gel, preventing suitable suctioning of the agarose along the capillary tube.</li>
	<li>Place each bulb-tube-needle assembly in a free Petri dish, and let the agarose gel inside the capillary tubes solidify for 5 min. Carefully pull the capillary tube with your hands out of the rubber stopper in the bulb dispenser cylinder, leaving the needle and the agarose gel in place inside the tube.</li>
	<li>Manually extract the acupuncture needle by slowly pulling it out of the capillary tube; the newly solidified agarose cylinder also slides out of the tube in this procedure, still surrounding the needle. Gently nudge the micro-column along the acupuncture needle with the tip of sterile forceps to move it to the end. Place the needle over an open, DPBS-containing Petri dish and push the micro-column into the DPBS with forceps. 
	NOTE: If the agarose micro-column remains within the glass capillary tube upon removal of the acupuncture needle, slowly push the agarose micro-column out of the capillary tube with a 25-mm gauge needle and into the dish with DPBS.</li>
	<li>Sterilize microscalpels or forceps using a hot bead sterilizer. Make 4% w/v agarose by weighing 4 g of agarose and transferring it to 100 mL of DPBS. Heat and stir as explained in step 1.2 to obtain a clear 4% liquid agarose solution. Maintain heating and stirring of the solution throughout the following steps.</li>
	<li>Transfer the Petri dishes containing the micro-columns to a dissection hood, and move a micro-column with fine forceps to an empty Petri dish. Utilize the stereoscope for visual guidance and a microscalpel to cut the micro-columns to the desired length. Trim both ends to form beveled ends at 45o angles from horizontal to facilitate handling of the micro-columns during extracellular matrix (ECM) and cell addition.</li>
	<li>Repeat the previous step for three more micro-columns in the same Petri dish and line up the four constructs in parallel with a separation of &#60;3 mm between each of the cylinders. Load 50 &#181;L of 4% agarose solution with a micropipette and pour a streak/line of liquid over the micro-column array to connect and bundle the constructs into groups of four (hereafter called &#34;micro-column boats&#34;). Avoid movement of 4% agarose to the ends of the micro-columns, which may clog the interior, by minimizing the distance between the constructs and adding the agarose quickly in a thin line. 
	NOTE: Arranging groups of four micro-columns into &#34;boats&#34; is not required to fabricate the astrocytic scaffolds. Nevertheless, the boats serve to hasten the fabrication process and to offer a safer way to move and handle micro-columns in later steps.</li>
	<li>Let the micro-column boat cool for 1-2 min to permit gelation of the added 4% agarose.Pick up the micro-column boat with fine forceps by the connecting 4% agarose, and move the micro-columns to the other DPBS-containing Petri dish prepared in step 1.1.3. Fabricate more boats with the remaining micro-columns as desired.</li>
	<li>Move the Petri dishes containing the micro-columns and/or boats to a biosafety cabinet and sterilize with exposure to ultraviolet (UV) light for 30 min. 
	Caution: Wear appropriate protection to prevent exposure to UV.</li>
	<li>Store the dishes with the micro-column boats in DPBS at 4 &#176;C, if ECM addition and cell plating will not occur immediately afterwards, up until 1 week. Repeat the micro-column fabrication steps if the constructs are not used during this timeframe.</li>
</ol>
2. Primary Cell Culture and Isolation
<ol>
	<li>Cortical astrocyte isolation from rat pups
	<ol>
		<li>In preparation for the following steps, add 20 mL of Hank&#39;s balanced salt solution (HBSS) to four 10 cm Petri dishes that will serve as reservoirs for the dissected tissues. Keep all the dishes on ice. Sterilize clean surgical scissors, forceps, micro-spatula, and micro-scalpels in a hot bead sterilizer.</li>
		<li>Prewarm 0.25% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.15 mg/mL deoxyribonuclease (DNase) I in HBSS at 37 &#176;C. In addition, prewarm the astrocyte culture medium at 37 &#176;C, which consists of Dulbecco&#39;s Modified Eagle Medium (DMEM) with Ham&#39;s F-12 Nutrient Mixture and 10% fetal bovine serum (FBS).</li>
		<li>Anesthetize postnatal day 0 or day 1 Sprague-Dawley rat pups by exposing them to hypothermic conditions and euthanize by decapitation. Pin the head into a stage using a 19 mm gauge needle placed in the snout anterior to the eyes.</li>
		<li>Make an incision with a scalpel down the middle posterior to anterior, from the base of the neck up to just behind the eyes. Carry out another incision going laterally from directly behind the eyes downwards, forming a T-shape. Use fine forceps to pull the cranial skin/skull off to the side.</li>
		<li>Hold the head by the snout (facing up) by placing one prong of the forceps in the mouth of the animal and the other on the outside surface. Remove the brain with a sterile micro-spatula and place it in one of the Petri dishes filled with chilled HBSS. Place this Petri dish on ice immediately afterwards and at all times except when in use.</li>
		<li>Situate a granite block previously stored at -20 &#176;C below a stereoscope inside a dissection hood. Place the Petri dish with the brain tissue on the surface of the granite block to preserve its low temperature during the dissection procedure. Use the stereoscope as visual guidance throughout the following steps.</li>
		<li>If the olfactory bulbs remain intact, extract them by cutting with forceps or microscalpels. In addition, use a microscalpel to remove the cerebellum and to make a midline incision separating the two cerebral hemispheres. Transfer the hemispheres with forceps to a Petri dish containing fresh, chilled HBSS.</li>
		<li>Dissect the midbrain structures from the inside of the hemispheres with a microscalpel to obtain only the separated cortices. Use fine forceps to peel the meninges, a sheet-like structure, off from the cortical tissue and to transfer the isolated cortices to a new Petri dish with cold HBSS. Use the microscalpel to mince the tissue in order to increase the surface area for trypsin action in the next step.</li>
		<li>Use a Pasteur pipette to transfer the cortices to a 15 mL centrifuge tube containing 4 mL of prewarmed trypsin-EDTA (8 cortices in each tube). Expose the cortical tissue to trypsin-EDTA for 5-7 min at 37 &#176;C.</li>
		<li>With a Pasteur pipette, carefully remove the trypsin-EDTA and add 400 &#181;l of 0.15 mg/mL DNAse I solution to the centrifuge tube. Agitate the tube or vortex until all the tissue is dissociated and there are no remaining tissue pieces in the liquid. If it is not possible to completely dissolve the tissue, remove the remaining fragments with the tip of a Pasteur pipette.</li>
		<li>Centrifuge the tube containing the dissociated cell solution at 200 x g for 3 min. Remove the supernatant with a Pasteur pipette without disturbing the cells. Add 1 mL of astrocyte culture medium (defined in step 2.1.2) to the tube with a micropipette and agitate to resuspend and create a homogeneous solution.</li>
		<li>Transfer 10 mL of astrocyte culture medium with a serological pipette to a T-75 flask. Add 250 &#181;l of the 1 mL cell solution (prepared in step 2.1.11) with a micropipette to the flask to plate one pup brain worth of cells per flask. Distribute the discharged cell solution evenly across the culture medium and gently agitate the flask to further promote an even distribution.</li>
		<li>Culture the plated flasks in a humidified incubator at 37 &#176;C and 5% CO2. After reaching 24 and 72 h in culture, mechanically agitate the flask to detach non-adherent cell types, such as neurons and oligodendrocytes.</li>
		<li>Afterwards, at each of these timepoints, perform a media change. Hold the flask vertically so the culture media lies on the bottom of the flask, not covering the adhered cells. Aspirate the culture medium with a Pasteur pipette, pressing the tip of the pipette against the bottom corner of the flask to avoid extracting the cells. Place the flask in the original horizontal position and gently add 10 mL of astrocyte medium over the cells with a serological pipette.</li>
		<li>Return the flasks to the incubator after each media change. After 90% confluency is achieved, mechanically agitate the flask once more to remove any remaining non-adhering cells.</li>
		<li>Passage the astrocytes by taking out the culture medium with a vacuum and a Pasteur pipette. Add 5 mL of 0.25% trypsin-EDTA with a serological pipette over the adhered cells. Gently agitate the flask to ensure the trypsin covers all the cells, and incubate the flask for 5-7 min at 37 &#176;C and 5% CO2.</li>
		<li>Quench trypsin by adding 1 mL of astrocyte medium to the cells with a serological pipette. Extract the cell solution from the flask with a serological pipette and transfer it to a sterile 15 mL centrifuge tube. Centrifuge the tube at 200 x g for 3 min.</li>
		<li>Remove the supernatant with a serological pipette and resuspend it in 1 mL of astrocyte culture medium. Agitate the tube to ensure the cell solution is homogeneous. Count the number of cells in the solution with a hemocytometer or an automatic cell counter. 
		NOTE: A flask that is 90% confluent typically yields approximately 3 million astrocytes.</li>
		<li>Add 10 mL of astrocyte culture medium to a T-75 flask. Perform a 1:4 dilution by transferring 250 &#181;l of the cell solution (step 2.1.18) with a micropipette to the T-75 flask already containing culture medium. Gently agitate to ensure a homogeneous cell distribution throughout the surface of the flask.</li>
		<li>Repeat steps 2.1.16-2.1.19 each time 90% confluency is achieved to passage the cells.</li>
	</ol>
	</li>
	<li>Cortical Neuron Isolation from Rat Fetuses
	<ol>
		<li>&#8203;Follow similar preparations as steps 2.1.1 and 2.1.2, with the exception that the prewarmed media is co-culture media, consisting of Neurobasal medium + 2% B-27 supplement (for neurons) + 1% G-5 serum-free supplement (for astrocytes) + 0.25% L-glutamine.</li>
		<li>Euthanize timed-pregnant embryonic day 18 Sprague-Dawley rats with carbon dioxide asphyxiation and confirm death by decapitation.</li>
		<li>Extract the rat fetuses and dissect the cortices from the rest of the cerebral tissue in Petri dishes containing HBSS on the surface of the chilled granite block, using a stereoscope for visual guidance and sterilized scissors, microscalpel, and forceps. After the successive dissection of the heads, brains, cerebral hemispheres, and cortices, transfer each tissue to a new HBSS-filled Petri dish.</li>
		<li>Mince the cortical tissue into smaller fragments to increase the surface area for trypsin. Transfer 4-6 cortices with a Pasteur pipette to a tube with 5 mL of prewarmed trypsin-EDTA and maintain at 37 &#176;C to dissociate the tissue. At 5-7 min manually agitate the tube to mix and prevent clumping of the tissue.</li>
		<li>Remove the tube from 37 &#176;C after 10 min. As explained previously in step 2.1, extract the trypsin-EDTA and substitute with 1.8 mL of 0.15 mg/mL DNAse solution. Afterwards, vortex the tissue until the solution appears homogeneous, with no tissue fragments floating in the liquid.</li>
		<li>Centrifuge the dissociated tissue solution at 200 x g for 3 min. After removing the supernatant, resuspend in 2 mL of co-culture medium. Count the number of cells in this solution with a hemocytometer or an automatic cell counter. 
		NOTE: The usual yield is 3.0-5.0 x 106&#160;cells/cortical hemisphere.</li>
		<li>Prepare a new cell solution with a density of 2.0-4.0 x 105&#160;cells/mL in the co-culture media defined above.</li>
	</ol>
	</li>
</ol>
3. Development of the Astrocytic Cables Inside the Micro-columns
<ol>
	<li>ECM core fabrication 
	NOTE: The ECM has to be added to the interior of the hydrogel micro-columns on the same day in which cell seeding will be performed.
	<ol>
		<li>Inside a biosafety cabinet, prepare a 1 mg/mL solution of rat tail type I collagen in co-culture medium in a sterile microcentrifuge tube. Maintain the microcentrifuge tube with the ECM solution on ice at all times except when in use.</li>
		<li>Transfer 1-2 &#181;L of the ECM solution with a micropipette onto a strip of litmus paper to estimate its pH. Add 1 &#181;L of 1 N sodium hydroxide (NaOH) or hydrochloric acid (HCl) with a micropipette to increase or decrease the pH of the ECM solution, respectively, and pipette up and down to homogenize. Verify the new pH with a litmus paper strip and add more acid or base, as needed, until the pH is stable in the 7.2-7.4 range.</li>
		<li>Move the Petri dishes from step 1.2 to a dissection hood, and transfer 4-5 micro-columns or a boat with sterile fine forceps to an empty, sterile 35 or 60 mm Petri dish. Using the stereoscope for guidance, situate the 10 &#181;L tip of a micropipette at one end of each micro-column and suction to empty the lumen of DPBS and air bubbles. Confirm the absence of air bubbles with the stereoscope to ensure that the ECM added in the next step can flow freely across the lumen.</li>
		<li>Charge a P10 micropipette with ECM solution, place the 10 &#181;L tip against one end of the hydrogel micro-columns, and deliver enough solution to fill the lumen (approximately 3-5 &#181;L), observing the entry of ECM with the stereoscope. Pipette a small reservoir (2-4 &#181;L) of ECM solution on either end of the micro-column. 
		NOTE: Always manage 4-5 micro-columns or one boat at a time to prevent prolonged drying of the constructs, as this may result in the crumpling of the micro-column structure. Completely dried micro-columns firmly attach to the surface of the Petri dishes, which prevents their utilization for cell seeding. Excessive amounts of co-culture media (as a hydration measure) cannot be added to the micro-columns because this may cause the ECM to flow out during the incubation period described below.</li>
		<li>Pipette co-culture media in a ring around the Petri dish to provide a humidity sink to prevent the columns from drying out during incubation. Incubate the Petri dish containing the ECM-containing micro-columns at 37 &#176;C and 5% CO2&#160;for 1 h to promote polymerization of collagen before adding the neurons and/or astrocytes. 
		NOTE: The ECM should form a layer coating the inner surface of the hollow lumen, rather than a solid ECM core encompassing the interior, both of which can be observed using the stereoscope. If the ECM forms a core, continue the incubation period until only the layer is left. With this layer formed, the astrocyte cell solution can fill the interior of the micro-column in the plating steps.</li>
		<li>During the incubation period, prepare the astrocyte cell solution (as described below).</li>
	</ol>
	</li>
	<li>Astrocyte and Neuron Seeding in the Micro-Columns
	<ol>
		<li>Passage the plated astrocytes (between the fourth and twelfth passage) as explained in steps 2.1.16-2.1.19. After counting the number of cells in the flask, prepare cell solution at a density of 9.0-12.0 x 105&#160;cells/mL solution suspended in cell-culture media.</li>
		<li>Using a stereoscope, place the tip of a P10 micropipette at one end of the micro-columns, and transfer sufficient cell solution (~5 &#181;L) into the lumen to completely fill it. As done above with the ECM, add small reservoirs of cell solution to both ends of each micro-column.</li>
		<li>Incubate the plated micro-columns on the Petri dishes at 37 &#176;C and 5% CO2&#160;for 1 h to promote the attachment of astrocytes to the ECM. If neurons will not be added to micro-columns, proceed to step 3.2.5.</li>
		<li>Following the initial incubation period, add 1-2 &#181;L of the cortical neuron solution obtained in step 2.2.7 into both ends of the micro-columns with a micropipette, while observing under the stereoscope. Ensure that sufficient media is present in the dishes to avoid drying, and incubate again for 40 min at 37 &#176;C and 5% CO2&#160;to allow for the adhesion of neurons. 
		NOTE: Cortical neuron solution can also be added 1-2 days after bundle formation, performing step 3.2.4 after carefully removing the culture media from the Petri dish with a micropipette.</li>
		<li>After the incubation period, carefully fill the Petri dishes containing the plated micro-columns with 3 or 6 mL of co-culture media for 35 or 60 mm Petri dishes, respectively. Maintain the plated micro-columns in culture at 37 &#176;C and 5% CO2&#160;to promote the self-assembly of the aligned astrocytic bundles, which should form a bundled, cable-like structure after 6-10 h.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acupuncture needle (300 &#181;m diameter)</td>
			<td>Lhasa Medical</td>
			<td>HS.30x40</td>
			<td>The diameter may be varied according to the desired size for the micro-column lumen.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher</td>
			<td>08772B</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Invitrogen</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene disposable serological pipet</td>
			<td>Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter glass capillary tube (701 &#181;m)</td>
			<td>Fisher</td>
			<td>21-170J</td>
			<td>The diameter may be varied according to the desired size for the micro-column shell.</td>
		</tr>
		<tr>
			<td>Microcap bulb dispenser</td>
			<td>Fisher</td>
			<td>21-170J</td>
			<td>Bulb comes with the microcap tubes.</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher</td>
			<td>SP88857200</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bar</td>
			<td>Fisher</td>
			<td>1451352</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Sigma</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm gauge needle</td>
			<td>Fisher</td>
			<td>14-826-49</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscalpel</td>
			<td>Roboz Surgical</td>
			<td>RS-6270</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Sigma</td>
			<td>Z378550-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-spatula</td>
			<td>Fisher</td>
			<td>S50821</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tail type I collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Maintain at 4 &#186;C and remove only when needed. Use ice to preserve its temperature when in use.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher</td>
			<td>02-681-256</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher</td>
			<td>SS2661</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher</td>
			<td>SA48-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Litmus paper</td>
			<td>Fisher</td>
			<td>09-876-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170112</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>Bovine pancreatic deoxyribonuclease (Dnase) I</td>
			<td>Sigma</td>
			<td>10104159001</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11195</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr>
			<td>Postnatal day 0 or day 1 Sprague Dawley rat pups</td>
			<td>Charles River</td>
			<td>Strain 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal embryonic neuron basal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td>Store at 4&#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>B-27 serum free supplement</td>
			<td>Invitrogen</td>
			<td>12587010</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr>
			<td>G5 astrocytic supplement</td>
			<td>Invitrogen</td>
			<td>17503012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley embryonic day 18 rats</td>
			<td>Charles River</td>
			<td>Strain 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Fisher</td>
			<td>22-042816</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>EMESCO</td>
			<td>1194-352099</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher</td>
			<td>05-413-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisher</td>
			<td>13 998 076</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Katiyar, K. S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55848">Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration.</a> J. Vis. Exp. (2018).
This video demonstrates a method to generate three-dimensional astrocyte scaffolds within hydrogel micro-columns. The inside of the microcolumns are coated with collagen. Astrocytes are introduced, which adhere to collagen and, with further incubation, self-assemble into a three-dimensional scaffold.

1. Development of the Agarose Hydrogel Micro-columns

	Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a ...

Take a culture dish containing a micro-column boat comprising hollow tubes of defined diameter.&#160;&#160;&#160;&#160;&#160;&#160;
The micro-columns, made of a hydrogel, are filled with a buffer to prevent drying.
Under a stereomicroscope, remove the buffer and add an ice-cold collagen solution, a component of the extracellular matrix.&#160;&#160;&#160;
Introduce a media along the dish&#39;s inner edge to maintain column hydration. Incubate under physiological conditions for the collagen to polymerize, forming a coating inside the micro-columns.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
Add astrocytes inside the micro-columns and incubate to promote astrocyte adhesion to collagen.
Next, fill the dish with the medium and maintain it under physiological conditions.&#160;&#160;&#160;&#160;&#160;&#160;&#160;
Due to the micro-columns&#39; narrow diameter, the astrocytes acquire a bipolar morphology with processes extending along the length.
Over time, the astrocytes self-assemble to form a three-dimensional scaffold containing dense bundles of astrocyte processes.
The hydrogel-encased astrocyte scaffold is ready for downstream assays.&#160;&#160;&#160;&#160;&#160;&#160;&#160;

20 Views. طريقة في المختبر لتوليد سقالات الخلايا النجمية داخل أعمدة الهيدروجيل الدقيقة

Video: طريقة في المختبر لتوليد سقالات الخلايا النجمية داخل أعمدة الهيدروجيل الدقيقة - Experiment

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

الطباعة الحيوية ثلاثية الأبعاد للخلايا الفلكية القشرية لمورين للأنسجة الهندسية الشبيهة بالعصبية

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

JoVE Journal

Bioengineering

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

تحليل في الحجم والشكل واتجاهية شبكات من أستروسيتيس إلى جانب

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

تصور مورفولوجيا الخلايا النجمية باستخدام الأيونتوبهوريس الأصفر لوسيفر

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

Transcript

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns