eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Stock medium preparation and stem cell recovery
<ol>
	<li>Prepare culture medium stock (DFHGPS) by combining the reagents in steps 1.1.1.-1.1.5.
	<ol>
		<li>Add 210 mL of Dulbecco&#39;s Modified Eagle Medium with high glucose and L-glutamine (D). Store it at 4 &#730;C.</li>
		<li>Add 70 mL of Ham&#39;s F12 Nutrient mix with L-glutamine supplement (F). Store it at 4 &#730;C.</li>
		<li>Add 4.2 mL of 15 mM HEPES Buffer (H) and store it at room temperature.</li>
		<li>Add 4.2 mL of 10% D-glucose Solution (G) and store it at room temperature.</li>
		<li>Add 2.88 mL of penicillin/streptomycin (PS) solution. Aliquot the stock solution into 3 mL aliquots and store the aliquots at -20 &#176;C until needed. 
		NOTE: The final concentration of penicillin in the medium will be 100 units/mL, and the concentration of streptomycin will be 100 &#181;g/mL. This medium will be referred to as DFHGPS for the remainder of the protocol and should be stored at 4 &#176;C for use within 1-2 weeks. If needed, DFHGPS may be scaled up proportionally.</li>
	</ol>
	</li>
	<li>The day before cells are recovered, coat a T75 flask with 5 mL of conditioned medium obtained from previous cultures of the hNSC cell line and leave it in a 37 &#176;C incubator with 8.5% carbon dioxide (CO2) overnight. 
	NOTE: If currently culturing hNSCs, save the medium that cells have been growing in during a medium change. This is a conditioned medium as it has been &#34;conditioned&#34; by the cells. The conditioned medium can be saved in a screw cap tube and stored at 4 &#176;C for approximately 7 days. Contact the corresponding author to receive hNSCs. Conditioned medium is preferred but not absolutely required, particularly when first starting the culture of hNSCs.</li>
	<li>On the day of recovery, warm 20 mL of DFHGPS in a 50 mL screw cap tube in a 37 &#176;C water bath for at least 10 min and keep it in the water bath until ready to use.</li>
	<li>In a separate 15 mL screw cap tube, warm 10 mL of DFHGPS in a 37 &#176;C water bath for at least 10 min and keep it in the water bath until ready to use in step 1.13.</li>
	<li>Retrieve a container of ice and all medium components (Table 1). Thaw all medium components on the ice and leave them on the ice while preparing for the medium change.</li>
	<li>Obtain 5 mL of conditioned medium stock (stored at 4 &#176;C) and keep it in a 37 &#176;C water bath until ready to use in step 1.14. See note after step 1.2 if there is no conditioned medium or current human fetal neural stem cell culture.</li>
	<li>Retrieve the 50 mL screw cap tube from step 1.3 and place it in the biosafety cabinet (BSC). Transfer 10 mL of DFHGPS into a new 15 mL screw cap tube, and then place the 50 mL tube containing the remaining 10 mL of DFHGPS medium back in the 37 &#176;C water bath.</li>
	<li>Obtain a cryo-vial of hNSCs stored in liquid nitrogen. See note after step 1.2 if there is no current human fetal neural stem cell culture. 
	NOTE: Human neural stem cells were originally derived from discarded human fetal brains12 and maintained in culture without genetic modifications10. 5 &#215; 106 cells should be thawed and plated on a T75 flask. Each cryo-vial should contain 5 &#215; 106 cells; therefore, one vial per flask is needed.</li>
	<li>Thaw a vial of cells in a 37 &#176;C water bath by inverting the vial every 10 s for approximately 1 min or until the ice is melted and the contents of the vial are completely liquid. Do not submerge the whole vial under water to avoid potential contamination.</li>
	<li>In the BSC, use a P1000 micro-pipette to aspirate the thawed cell solution and add it drop-wise to the 15 mL tube from step 1.7, containing 10 mL of DFHGPS. To add the thawed cell solution drop-wise, slowly press down on the plunger so that only a drop or two is released at once. Meanwhile, swirl the 15 mL tube to allow a quick mixture.</li>
	<li>Centrifuge the cell suspension at 200 x g for 5 min at room temperature. Discard the supernatant, making sure to retain the cell pellet.</li>
	<li>During step 1.11, retrieve the 50 mL tube containing the remaining 10 mL from step 1.7. After the centrifugation, resuspend the cell pellet in the 10 mL DFHGPS and centrifuge again at 200 x g for 5 min at room temperature.</li>
	<li>During the final spin, prepare the new growth medium by following Table 1 to add growth factors to the 10 mL of DFHGPS medium from step 1.4 (Table 1) in BSC. Leave the new medium containing growth factors in the BSC until ready to use.</li>
	<li>Retrieve the T75 flask from step 1.2 and discard the conditioned medium coating the flask by aspiration. Then, add the 5 mL of conditioned media from step 1.6. to the flask using a serological pipette. Be careful not to scratch the bottom of the flask.</li>
	<li>Retrieve the tube of cells from the centrifuge and discard the supernatant by aspirating it, leaving the cell pellet intact.</li>
	<li>Resuspend the cell pellet in 10 mL of new media from step 1.13 by using a 5 mL serological pipette and pipetting up and down several times. Add the suspension to the coated T75 flask from step 1.14. Use the remaining 5 mL to rinse the tube that contained the cell pellet, and then add those 5 mL to the flask as well.</li>
	<li>Rock the T75 flask back and forth 3 times to ensure the cells are evenly distributed across the flask. Make sure the cap of the flask is loose to allow airflow. Under a light microscope, observe the cells, make notes, and take images if possible. 
	NOTE: The cells should look translucent and spherical. Make a note if there are cells that look dark, opaque, or have asymmetrical borders, as this can indicate cell death. Also, watch for any fungal or bacterial contamination, such as the media becoming yellow in color.</li>
	<li>Place the flask of cells in a 37 &#176;C incubator with 8.5% CO2. 
	NOTE: Use 8.5% instead of 5.0% CO2 to maintain the pH of this serum-free culture media in the range of 7.1-7.4.</li>
</ol>
2. hNSC Medium Change
NOTE: Change medium every 3-4 days.
<ol>
	<li>Turn on the BSC and clean thoroughly with 70% ethanol. Observe cells under a light microscope. 
	NOTE: Make notes on the appearance of the cells and sphere sizes. Three days after recovery or passage, cells will cluster together and begin to form non-adherent neurospheres. 
	CAUTION: If cells adhere to the bottom of the flask, the culture will need to be discarded as cell adherence will increase premature differentiation, and cells will be unable to maintain a stem state. If there are too many cells in the flask, the cells may form large spheres (greater than 2 mm in diameter), in which case, cells in the center of the sphere will begin to differentiate due to lack of access to growth factors in the medium. If spheres larger than 1 mm in diameter are observed, cells can be passaged (steps 3.1-3.22).</li>
	<li>Follow steps 1.4 and 1.5, and prepare 10 mL of fresh medium (see step 1.13).</li>
	<li>Tilt the flask to the side, allowing spheres to sink to the bottom of the flask.</li>
	<li>Remove 5 mL of conditioned media from the top of the pooled medium using a 5 mL serological pipette, taking care not to aspirate any neurospheres. Place the 5 mL of conditioned media into a clean 15 mL tube.</li>
	<li>Remove an additional 5 mL of conditioned media from the flask, again carefully avoiding the spheres, and place the 5 mL into the same 15 mL tube.</li>
	<li>Add 10 mL of new media from step 2.2 to the remaining 5 mL of conditioned media and cells in the flask. Set the flask down flat and observe cells under a light microscope.</li>
	<li>If it appears cells have been aspirated during the collection of the conditioned medium, spin the conditioned medium at 100 x g for 5 min at room temperature. Re-suspend any cells that accumulate at the bottom in 1 mL of the conditioned medium, and then add them to the culture flask.</li>
	<li>Store the 10 mL of unused conditioned media from steps 2.5/2.6 at 4 &#730;C in a 15 mL screw cap tube.</li>
	<li>Repeat steps 1.17/1.18.</li>
</ol>
3. hNSC Passage
<ol>
	<li>If expanding the NSCs into a new flask, make sure all new flasks are coated as in step 1.2.</li>
	<li>Clean the BSC as in step 2.1, and conduct steps 1.4 and 1.5.</li>
	<li>Prepare medium as in step 1.13. Each T75 flask requires 10 mL of medium. For multiple T75 flasks, multiply 10 mL of media by the number of flasks.</li>
	<li>Prepare either 1 or 3 mL of Dulbecco&#39;s phosphate-buffered saline (dPBS) and glucose and place in a 37 &#176;C water bath to warm for 10 min (for trypsin solution) according to the volumes in Table 2. Do not add trypsin or DNase at this time. DNase may be required to break down any DNA released from the dead cells due to mechanical or chemical cell dissociation. 
	NOTE: For one T75 flask passaged after 9-10 days of growth, there will be approximately 30 &#215; 106 cells if 5 &#215; 106 were originally seeded on the flask. Typically, 1 mL of dPBS solution is used for 10 &#215; 106 cells. Therefore, 3 mL of dPBS solution is needed for a T75 flask passaged after 9-10 days.</li>
	<li>Place a clean small weigh boat and hemocytometer in the hood. Clean with 70% ethanol and allow to air dry in the hood for approximately 5 min. 
	NOTE: The weigh boat will be used in steps 3.17.1.</li>
	<li>Retrieve the flask of cells that will be passaged from the incubator and bring into the BSC. Tilt flask gently allowing cells to sink to the bottom of the pooled media. There is about 15 mL of media in the flask.</li>
	<li>Remove about 10 mL of media in 5 mL portions (i.e. 5 mL + 5 mL). Place this 10 mL of media into a clean 15 mL tube labeled &#34;CM&#34; for &#34;conditioned medium&#34;. Be careful not to aspirate any of the cells settled in the remaining 5 mL of media in the flask; these cells and 5 mL of media will be subjected to step 3.8.
	<ol>
		<li>Take 3 mL of conditioned media from the &#34;CM&#34; tube (from step 3.7) and place into a 15 mL tube labeled &#34;trypsin inhibitor solution&#34;. This will be used in step 3.12. After removal of the 3 mL, there is 7 mL of conditioned media left in the &#34;CM&#34; tube. Set the &#34;CM&#34; tube aside until step 3.9.</li>
	</ol>
	</li>
	<li>Remove the 5 mL of media and cells remaining in the T75 flask and place in a clean 15 mL tube labeled &#34;Cells&#34;.</li>
	<li>Take the &#34;CM&#34; tube containing 7 mL of conditioned media (from step 3.7.1) and rinse the bottom of the T75 flask twice with 3.5 mL portions of conditioned media using a 5 mL serological pipette. After each rinse, place the 3.5 mL portions into the &#34;Cells&#34; tube. The purpose of this step is to remove all neurospheres (cells) from the T75 flask.</li>
	<li>Centrifuge the &#34;Cells&#34; tube at 100 x g for 5 min at room temperature. While cells are spinning, obtain dPBS/glucose solution from 37 &#176;C water bath and add the appropriate amount of trypsin and DNase according to Table 2.</li>
	<li>After the &#34;Cells&#34; tube has stopped spinning, remove all the conditioned medium supernatant and transfer to the &#34;CM&#34; tube.</li>
	<li>Take the tube labeled &#34;trypsin inhibitor solution&#34; from step 3.7.1. and add the volume of trypsin inhibitor indicated in Table 3. Use the same volume of trypsin inhibitor solution as was used for the trypsin solution.</li>
	<li>Place trypsin inhibitor solution in the 37 &#176;C water bath until needed.</li>
	<li>Add trypsin solution to the cell pellet in the 15 mL tube and pipette up and down approximately 5-10 times with a 5 mL pipette. Close the cap of the tube and incubate in a 37 &#176;C water bath for 5 min. After 5 min, retrieve the cells and pipette up and down 5-10 times. Then, incubate in the 37 &#176;C water bath for an additional 10-15 min.</li>
	<li>In the last 5 min, move the trypsin inhibitor solution to the BSC.</li>
	<li>Retrieve the cells after step 2.14 is complete and pipette the cells up and down until spheres are completely dissociated (20-30 times). Then, immediately add the trypsin inhibitor solution and pipette up and down 10 times. This solution of dissociated cells and trypsin inhibitor will be referred to as &#34;cell suspension&#34;.</li>
	<li>Count the number of cells in the cell suspension by following steps.
	<ol>
		<li>Pipette 15 &#181;L of Trypan Blue pre-mixture (containing 5 &#181;L of 0.4% Trypan Blue and 10 &#181;L of dPBS) onto a small weigh boat, and then add 5 &#181;L of the cell suspension. Pipette up and down 3-5 times to mix. Then, pipette 10 &#181;L of the Trypan Blue/cell suspension onto either side of a hemocytometer covered with a glass coverslip.</li>
		<li>Observe the cells under a light microscope and count the total number of cells in each of the four corner grids. Add these numbers together.</li>
	</ol>
	</li>
	<li>Repeat counting for the other side and average the two sides together.</li>
	<li>Multiply this number by 10,000 to give the total number of cells per mL. Multiply this number by the total number of milliliters to calculate the total number of cells in the cell suspension. Calculate how much volume of cell suspension will be needed to seed 5 million cells per T75 flask. 
	NOTE: Typically, after 9-10 days, a T75 will have 25-30 &#215; 106 cells. Therefore, if passaging all cells into new flasks, a total of 5-6 flasks is needed.</li>
	<li>Add the volume of cell suspension calculated in step 3.19 to each T75 flask to obtain 5 &#215; 106 cells per flask. If the volume is less than 5 mL, add additional condition medium to make up to a total of 5 mL. Then add 10 mL of new DFHGFPS media containing growth factors (Table 1) to the flask.</li>
	<li>Repeat steps 1.17 and 1.18 and ensure the flask is labeled with the type of cells, the dates, the number of cells in the flask, and the passage number (this is the number of times the cell has been passaged). Then, move to Step 4.</li>
	<li>If needed, seed extra cells into one T75 flask at a density of up to 30 &#215; 106 cells per flask overnight and freeze the next day according to Step 4.</li>
</ol>
4. hNSC Freezing and Storage
<ol>
	<li>The day after passaging, retrieve the flask from the incubator containing the cells for freezing (from steps 3.19 and 3.22). Tilt the flask and remove all medium and cells from the flask, and place in a 15 mL tube. Then, centrifuge the tube at 216 x g for 5 min.</li>
	<li>During the centrifugation process, prepare the freezing medium (Table 4). For every 5 &#215; 106 cells, prepare 1 mL of freezing medium. The number of cells was determined in step 3.19 when determining how many cells to put in each flask. This number will not have changed overnight.</li>
	<li>Prepare cryo-preserve vials with labels of cell name, passage number, cell number, initials and date. After centrifugation, remove supernatant by aspiration and re-suspend cell pellet in freezing medium so that there are 5 &#215; 106 cells/mL. Using a 5 mL pipette, aliquot 1 mL per vial and seal tightly (5 &#215; 106 cells per vial).</li>
	<li>Place vials in a cryo-preserve container with 250 mL of isopropanol (the maximum usage is 5 times per replacement of isopropanol). Store in a -80 &#176;C freezer overnight, and then transfer to a liquid nitrogen tank storage system.</li>
</ol>
5. Plating adherent hNSC for validation
<ol>
	<li>The day before passaging (steps 3.1-3.22), clean the BSC as in step 2.2 and obtain a 24-well plate and sterile glass 12 mm coverslips.</li>
	<li>Using forceps, place a single coverslip on the bottom of each well of the plate.</li>
	<li>Coat the wells containing coverslips with 0.01% poly-D-lysine (PDL) in sterile water and incubate for 1 h at 37 &#730;C. For a 24-well plate, use 250 &#956;L of PDL per well.</li>
	<li>Remove the PDL from the wells, and then coat it with 1 &#181;g/cm2 laminin/dPBS (250 &#956;L per well). Incubate the plate overnight at 37 &#176;C. If not needed the next day, seal the plate with parafilm by wrapping the parafilm around the edges of the plate and store at 4 &#176;C. 
	NOTE: Plates coated with laminin can be stored for up to 2 weeks if sealed with parafilm as long as coverslips do not dry.</li>
	<li>Passage cells as described in steps 3.1-3.22, and then determine the number of cells to seed into the 24-well plate with coated coverslips.</li>
	<li>Remove any excess laminin solution from wells through aspiration, and rinse once with 0.5 mL of dPBS. Then, seed cells into the wells at a density of 0.6-1 x 105 cells/cm2 per well. Use any remaining cells according to steps 3.16-3.21.</li>
	<li>At various times during the 24-well plate culture, fix and stain the cells for various stem cell markers.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (10%)</td>
			<td>Sigma</td>
			<td>G8644</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Corning/cellgro</td>
			<td>25-060-c1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep (100x)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I4011</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fibroblast growth factor</td>
			<td>R&#38;D system</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>R&#38;D system</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>leukemia inhibitory factor</td>
			<td>R&#38;D system</td>
			<td>7734-LF</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine hydrobromide(PDL)</td>
			<td>Sigma</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning/cellgro</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4378-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Forma</td>
			<td>Model# 3110</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo fisher Scientific</td>
			<td>75004221</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Forma scientific Claas II A/B3</td>
			<td>Model# 1284</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80 &#176;C)</td>
			<td>Forma scientific,Inc</td>
			<td>model# 8516</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon TE2000-E microscope with C1si confocal system</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiating human neural stem cells into neural and astrocyte progenitors

Source: McGrath, E. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018).
This video demonstrates the method to differentiate human neural stem cells from neurospheres into neural and astrocyte progenitors using enzymatic treatment and culturing in a growth factor-rich medium, which eventually leads to maturation into specialized progenitor cells.

Table 1: Growth factors for New Proliferation Medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DFHGPS media for one T75 </td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>TPPS*</td>
			<td>173 &#181;L</td>
		</tr>
		<tr>
			<td>200 mM L-Glutamin</td>
			<td>50 &#181;L</td>
		</tr>
		<tr>
			<td>10 mg/mL Insulin**</td>
			<td>25 &#181;L</td>
		</tr>
		<tr>
			<td>20 &#181;g/mL Epidermal growth factor</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>20 &#181;g/mL Basic fibroblast growth factor</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>10 &#181;g/mL Leukemia inhibitor factor</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>5 mg/mL Heparin</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td colspan="2">* Mixture containing 100 &#181;g/mL Transferrin, 100 &#181;M putresine, 20 nM progesterone, and 30 nM sodium selenite.&#160; The mixture is made from concentrated stocks, and aliquots are stored at -80 &#176;C and preferablly used within 6 weeks preparation.&#160;&#160; The concentrated stocks are 10 mg/mL transferrin, 30 mM putrescine, 10 &#181;M progesterone and 15 &#181;M sodium selenite stored at -80 &#730;C.</td>
		</tr>
		<tr>
			<td colspan="2">** Insulin is dissolved in 0.01 N hydroen chloride, filtered through&#160; 0.2 &#181;M low protein binding filter, and stored at 4 &#730;C for up to 6 weeks.</td>
		</tr>
	</tbody>
</table>
Table 2: Preparation of trypsin.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>dPBS*</td>
			<td>1 mLa</td>
			<td>3 mLb</td>
		</tr>
		<tr>
			<td>10% glucose</td>
			<td>60 &#181;L</td>
			<td>180 &#181;L</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>10 &#181;L</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>Dnase**</td>
			<td>5 &#181;L</td>
			<td>15 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">* Dulbecco&#39;s phosphate-buffered saline</td>
		</tr>
		<tr>
			<td colspan="2">** Deoxyribonuclease</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">a for 10 million cells</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">b for 30 million cells</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3: Preparation of trypsin inhibitor.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Conditioned media</td>
			<td>1 mLa</td>
			<td>3 mLb</td>
		</tr>
		<tr>
			<td>Trypsin Inhibitor</td>
			<td>10 &#181;L</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>a for 10 million cells</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>b for 30 million cells</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 4: Preparation of freezing medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DFHGPS*</td>
			<td>0.7 mLa</td>
			<td>2.1 mLb</td>
		</tr>
		<tr>
			<td>FBS** 20%</td>
			<td>0.2 mL</td>
			<td>0.6 mL</td>
		</tr>
		<tr>
			<td>DMSO*** 10%</td>
			<td>0.1 mL</td>
			<td>0.3 mL</td>
		</tr>
		<tr>
			<td colspan="3">* DMEM, F12, HEPES, glucose, penicillin-streptomycin</td>
		</tr>
		<tr>
			<td colspan="2">** Fetal bovine serum</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">*** Dimethyl sulfoxide</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">a for 5 million cells in one vial</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">b for three vials, 5 million cells/vial</td>
		</tr>
	</tbody>
</table>

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Stock medium preparation and stem ...

Begin with non-adherent neurospheres, a three-dimensional aggregate of human neural stem cells in a cell culture-derived conditioned medium.
This medium contains nutrients, growth factors, and extracellular matrix components that maintain cell viability.
Centrifuge to collect the neurospheres.
Treat them with trypsin and DNase enzymes, mix the contents, and then incubate.
Trypsin disrupts cell-to-cell adhesion and facilitates cell detachment, while DNase degrades free DNA to prevent cell clumping.
Pipette the contents repeatedly to form a single-cell suspension. Then, add a trypsin inhibitor to neutralize the enzyme activity.
Centrifuge and remove the supernatant, and then resuspend the cells in a proliferation medium containing neural growth factors.
Seed the cell suspension onto a polymer-coated coverslip placed in a multi-well plate. Incubate to allow the stem cell attachment to the polymer.
Further, based on the absorption of specific growth factors, the stem cells differentiate into neural and astrocyte progenitors, which are ready for further maturation into specialized cell types.

differentiating-human-neural-stem-cells-into-neural-astrocyte

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

36 조회수. 출처: McGrath, E. L., et al. 면역세포화학 분석을 위한 배양된 인간 태아 뇌 신경 줄기세포의 지카 바이러스 감염. J. Vis. 특급 (2018). 이 동영상은 효소 처리를 사용하여 인간의 신경 줄기세포를 신경 및 성상세포 전구세포로 분화시키고 성장 인자가 풍부한 배지에서 배양하여 결국 특수 전구 세포로 성숙시키는 방법을 보여줍니다. 

비디오: 인간 신경 줄기 세포를 신경 및 성상 세포 전구 세포로 분화 - 개념

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

말초 혈액 유래 인간 유래 다 능성 줄기 세포에서 연골 세포 분화

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

연구

JoVE Journal

발생학

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

인간의 신경의 집계의 cGMP-해당 확장 방법은 줄기 및 전구 세포는 다 능성 줄기 세포 또는 태아 뇌 조직에서 파생 된

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

신경과학

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

마우스 배아 줄기 세포에서 신경 전조및 뉴런의 분화 및 특성화

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

내레이션 대본

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors