developing human ipsc-derived motor nerve organoids using a microfluidic chip

Developing Human iPSC-Derived Motor Nerve Organoids Using a Microfluidic Chip

To induce 3D iPS cell differentiation into motor neurons, seed 4 x 104 iPS cells per well into the appropriate number of wells of a 96-well U-bottom plate in 100 microliters of feeder and serum-free cell culture medium supplemented with 10 micromolar Y27632.
The next day, replace the supernatant in each well with 100 microliters of KSR medium supplemented with 10 micromolar SB-431542 and 100 nanomolar LDN-193189. On days 2 and 3, replace the supernatants with 100 microliters of KSR medium supplemented with 10 micromolar SB-431542, 100 nanomolar LDN-193189, 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and one micromolar smoothened agonist. On days 4 and 5, replace the supernatants with a mixture of 75% of KSR medium and 25% of N2 medium, supplemented with 10 micromolar SP-431542, 100 nanomolar LDN-193189, 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist.
On days 6 and 7, replace the supernatants with a mixture of 50% of KSR medium and 50% of N2 medium, supplemented with 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist. On days 8 and 9, replace the supernatants with a mixture of 25% of KSR medium and 75% of N2 medium, supplemented with 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist. On days 10 and 11, replace the supernatants with N2 medium supplemented with 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist. On day 12, replace the supernatants in each well with 100 microliters of maturation medium, supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor per well.
To induce motor nerve organoid formation, replace the coating solution in the PDMS chip with 150 microliters of maturation medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor, and use a wide-bore micropipette tip to gently add motor neuron spheroids from one well of the 96-well plate into the inlet of the microchannel of the chip. Place a small reservoir of sterile water near the tissue culture chip in the dish to prevent medium evaporation, and place the dish in a 37 degrees Celsius and 5% carbon dioxide incubator.
Every two to three days, replace half of the exhausted culture medium from the center of the medium reservoir with fresh maturation medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor. Axons will grow from the motor neuron spheroid into the channel, spontaneously assembling into a single bundle over a period of two to three weeks to form a motor nerve organoid.

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1. SU-8 mold fabrication by photolithography
NOTE: This procedure involves hazardous chemicals. Use fume hood and PPE throughout.
<ol>
	<li>Clean the silicon wafer (4-inch in diameter, 1-mm thickness, polished) with acetone and blow with nitrogen gas. Then bake at 180 &#176;C for 3 min to dry.</li>
	<li>Dispense 3 mL of SU-8 2100 on to a cleaned wafer.</li>
	<li>Coat the SU-8 uniformly on the wafer using a spin coater at 500 rpm for 10 s and then, sequentially spin at 1500 rpm for 30 s with an acceleration of 300 rpm/s to obtain a 150 &#181;m thick layer of SU-8. 
	NOTE: Make sure that the silicon wafer is positioned at the center of the spin coater and correctly fixed by vacuum.</li>
	<li>Soft bake the wafer on the hot plate at 50 &#176;C for 10 min, at 65 &#176;C for 7 min, and at 95 &#176;C for 45 min.</li>
	<li>Set the photomask (Figure 1) to the mask aligner and expose UV light (365 nm) for 60 s. 
	NOTE: Exposure time needs to be optimized by an appropriate dose of UV light.</li>
	<li>After exposure, bake the wafer at 65 &#176;C for 6 min, and at 95 &#176;C for 13 min on the hot plate.</li>
	<li>Develop the wafer for 10-20 min in SU-8 developer with agitation using an orbital shaker, changing the developing solution once during the process. 
	NOTE: Extend the developing time when the debris of SU-8 remains.</li>
	<li>Rinse the wafer in isopropanol and gently dry the wafer with nitrogen gas.</li>
	<li>Measure the height of the deposited SU-8 by a measuring microscope and make sure it is approximately 150 &#181;m thick. It can be stored indefinitely at room temperature.</li>
</ol>
2. Polydimethylsiloxane (PDMS) microfluidic-based tissue culture chip fabrication
<ol>
	<li>Fix the SU-8-deposited wafer to a container (e.g., 15 cm plastic Petri dish) by double-sided tape.</li>
	<li>Blow the dust off the wafer using nitrogen gas.</li>
	<li>To silanize, put the SU-8-deposited wafer in a vacuum chamber together with a small container (e.g., 35 mm dish). Drop 10 &#181;L of (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane into the small container. Do not directly apply the (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane to SU-8 wafer.</li>
	<li>Close the vacuum chamber tightly and turn on a vacuum pump for at least 2 h.</li>
	<li>Take a plastic cup and pour the silicone elastomer (e.g., Silpot 184 or equivalently, Sylgard 184) and the curing agent at a 10:1 weight ratio. Then, mix well using a spatula and degas in the vacuum chamber until bubbles are removed entirely.</li>
	<li>Pour the PDMS mixture into the container with the SU-8 wafer to the desired thickness (3-4 mm) and degas again to remove bubbles.</li>
	<li>Bake the PDMS in an oven at 60 &#176;C for at least 3 hours to fully cure the PDMS.</li>
	<li>After cooling, cut off the cured PDMS from the wafer by using a scalpel or a razor blade.</li>
	<li>To create the two chambers of the tissue culture chip, punch two holes where the two compartments are located by using a 1.5 mm diameter biopsy punch.</li>
	<li>To create a medium reservoir, prepare another PDMS mixture (silicone elastomer and the curing agent at a 10:1 weight ratio) and pour it into a new 10 cm Petri dish. Adjust the pouring volume to 5 mm of thickness of PDMS.</li>
	<li>Bake the PDMS in an oven at 60 &#176;C for at least 3 hours to fully cure the PDMS.</li>
	<li>After cooling the PDMS, cut off the cured PDMS with a scalpel to get a rectangular ring.</li>
	<li>Bond the bottom layer with the medium reservoir by applying uncured PDMS between them and baking the assembled PDMS layers. This bonded structure results in the PDMS tissue culture chip.</li>
	<li>Clean the PDMS tissue culture chip by scotch tape to remove dust and small particles from the surface. PDMS tissue culture chips can be stored at room temperature if protected from dust and UV.</li>
</ol>
3. Preparation of culture
<ol>
	<li>Culture medium 
	NOTE: All media listed below should be filtered for sterilization unless otherwise stated. Prepared media may be stored at 4 &#176;C and used within a month.
	<ol>
		<li>To prepare mTeSR Plus medium: combine one bottle of 100 mL mTeSR Plus 5x supplement with one bottle of 400 mL of mTeSR Plus Basal Medium.</li>
		<li>To prepare 100 mL of KSR medium: In 85 mL of Dulbecco&#39;s Modified Eagle Medium or DMEM/F12, add 15 mL of Knockout serum replacement (KSR, 15%), 1 mL of commercial glutamine supplement (1%) and 1 mL of non-essential amino acid (NEAA, 1%).</li>
		<li>To prepare 100 mL of N2 medium: In 100 mL of Neurobasal medium, add 1 mL of N2 (100x), 1 mL of commercial glutamine supplement, and 1 mL of NEAA.</li>
		<li>To prepare 250 mL of Maturation medium: In 250 mL of Neurobasal medium, add 5 mL of B27 (2%), 2.5 mL of commercial glutamine supplement (1%), and 2.5 mL of Penicillin/Streptomycin (1%).</li>
		<li>Resuspend the compounds (retinoic acid (RA), SB431542, LDN-193189, SU5402, DAPT, SAG, Y-27632) in cell-culture-grade DMSO to the desired concentration. Prepare aliquots and store them at -20 &#176;C for up to 6 months. The following stock solutions are used: 1 mM RA, 10 mM SB431542, 100 &#956;M LDN-193189, 10 mM SU5402, 10 mM DAPT, 1 mM SAG, 10 mM Y-27632.</li>
	</ol>
	</li>
	<li>Coating 
	NOTE: To prevent polymerization of the basement membrane matrix by heat, avoid repetitive freeze-thaw cycles. Handle all coating procedures with pre-cooled pipette tips and tubes if possible. The basement membrane matrix should be thawed overnight at 4 &#176;C and aliquoted using pre-chilled pipette tips and tubes. The aliquots can be frozen at -20 &#176;C or -80 &#176;C.
	<ol>
		<li>Thaw the frozen aliquot at 4&#176;C on ice. The aliquot should be kept cold during the coating procedure. Using a pre-cooled pipette tip, dilute the basement membrane matrix with ice-cold DMEM/F12 at a ratio of 1:40. Unused diluted basement membrane matrix can be stored at 4 &#176;C for 2-3 days, given that no polymerization occurred.</li>
		<li>Add 1 mL of the basement membrane matrix/DMEM-F12 solution to coat one well of the 6-well plate.</li>
		<li>Incubate the plate at room temperature for at least an hour, or 4 &#176;C overnight. Coated plates can be stored at 4 &#176;C for a maximum of one week.</li>
	</ol>
	</li>
</ol>
4. Maintenance of iPS cells
<ol>
	<li>Prepare basement membrane matrix-coated dishes as previously mentioned in step 3.2.</li>
	<li>Fully aspirate the mTeSR Plus medium. Wash the well once with PBS and add 0.5 mL of passaging reagent (see Table of Materials). Wait a few seconds and aspirate the solution.</li>
	<li>Incubate the plate at 37 &#176;C in the incubator for 5 min or until cells become round. 
	NOTE: Incubation time might vary between different iPS cell lines and the confluency. Please check periodically under the microscope to determine the dissociation time during the incubation.</li>
	<li>Add 1 mL of mTeSR Plus medium and tap the plate for 30-60 s to detach the colonies.</li>
	<li>Gently mix 1 mL of the cell suspension solution with 7 mL of fresh mTeSR plus medium. Do not pipette more than 5 times.</li>
	<li>Plate at a ratio of 1:8. Typically add 1 mL of the suspension from step 5.5 and add 1 mL of mTeSR plus media supplemented with 5-10 &#956;M of Y-27632 (ROCK inhibitor). Passage dilution ratio depends on iPSC line.</li>
	<li>Place the cell in a 5% CO2/37 &#176;C incubator. On the next day, remove Y-27632 by adding fresh mTeSR Plus medium. Thereafter, change media every other day initially, and every day as the cell reaches higher confluency.</li>
</ol>
5. Differentiation of iPS cells into motor neurons
<ol>
	<li>Passaging iPSC for motor neuron differentiation 
	NOTE: Differentiation can be successfully conducted in 3D (5.2) protocol.
	<ol>
		<li>Allow undifferentiated iPS cells to grow until they reach a confluency of approximately 80% in mTeSR Plus medium in a 6-well plate.</li>
		<li>Completely aspirate the medium. Immediately wash the well once with sterile PBS and add 0.5 mL of cell dissociation solution to the cells.</li>
		<li>Incubate the plate at 37 &#176;C in the incubator for approximately 2-3 min, or until cells become separated and round but remain attached to the well.</li>
		<li>Add 1 mL of medium and gently pipette up and down a few times using a 5 mL serological pipette. Transfer the cell suspension to a 15 mL tube consisting of 4 mL of the medium.</li>
		<li>Centrifuge at 200 x g for 3 min.</li>
		<li>Carefully aspirate the supernatant, leaving the pellet undisturbed, and resuspend the cells in 1 mL of the medium supplemented with 10 &#181;M of Y-27632.</li>
		<li>Count the cells using a hemocytometer and proceed to either 5.2 (3D differentiation).</li>
	</ol>
	</li>
	<li>Formation of motor neuron spheroid (MNS) in 3D differentiation (&#34;3D protocol&#34;) 
	NOTE: A complete medium change is done daily from Days 0-12 of differentiation (Figure 2).
	<ol>
		<li>Seed the iPS cells from step 5.1.7 to a 96 well U bottom plate at 40,000 cells/well in 100 &#181;L of mTeSR Plus supplemented with 10 &#181;M of Y-27632.</li>
		<li>On the next day, replace each well with 100 &#181;L of the fresh medium.</li>
		<li>On days 0 and 1: Aspirate the culture medium and replace with 100 &#181;L of KSR medium (3.1.2) supplemented with 10 &#181;M SB431542 and 100 nM LDN-193189.</li>
		<li>On days 2 and 3: Aspirate the culture medium and replace with 100 &#181;L of KSR medium supplemented with 10 &#181;M SB431542, 100 nM LDN-193189, 5 &#181;M DAPT, 5 &#181;M SU5402, 1 &#181;M RA and 1 &#181;M SAG.</li>
		<li>On days 4 and 5: Prepare a mixed medium consisting of 75% KSR medium and 25% N2 medium (3.1.3). Then, aspirate the culture medium and replace with 100 &#181;L of mixed medium supplemented with 10 &#181;M SB431542, 100 nM LDN-193189, 5 &#181;M DAPT, 5 &#181;M SU5402, 1 &#181;M RA, and 1 &#181;M SAG.</li>
		<li>On days 6 and 7: Prepare a mixed medium consisting of 50% KSR medium and 50% N2 medium. Then, aspirate culture medium and replace it with 100 &#181;L of mixed medium supplemented with 5 &#181;M DAPT, 5 &#181;M SU5402, 1 &#181;M retinoic acid and 1 &#181;M SAG.</li>
		<li>On days 8 and 9: Prepare a mixed medium consisting of 25% KSR medium and 75% N2 medium. Then, aspirate culture medium and replace with 100 &#181;L of mixed medium supplemented with 5 &#181;M DAPT, 5 &#181;M SU5402, 1 &#181;M RA and 1 &#181;M SAG.</li>
		<li>On days 10 and 11: Replace medium with 100 &#181;l of N2 medium supplemented with 5 &#181;M DAPT, 5 &#181;M SU5402, 1 &#181;M RA and 1 &#181;M SAG.</li>
		<li>On day 12: Proceed to transfer the MNs into the tissue culture chip (Step 6) or replace medium with 100 &#181;L of the Maturation medium (3.1.4) supplemented with 20 ng/mL brain-derived neurotrophic factor (BDNF). 
		NOTE: The MNS can be transferred to the tissue culture chip starting from day 12 until day 19. Spheroids that are not transferred should be kept cultured in 96 well U bottom plates in Maturation medium supplemented with 20 ng/mL BDNF until transfer.</li>
	</ol>
	</li>
</ol>
6. Preparation of the tissue culture chip for motor nerve organoid (MNO) formation
<ol>
	<li>Sterilize the prepared PDMS (from step 2.13) by immersing it in 70% ethanol in the Petri dish for at least 1 h. 
	NOTE: All following steps should be manipulated in a biosafety cabinet.</li>
	<li>Sterilize the microscope glass (76 x 52 mm) by immersing it in 70% ethanol in the Petri dish.</li>
	<li>During the drying process of the microscope glass, place the PDMS device on the half wet-microscope glass and let it dry completely by waiting overnight. Once it is completely dried, the PDMS device should adhere to the glass. 
	NOTE: This bonding is not permanent to allow detaching of the PDMS devices from the microscope glass after the culture for tissue collection. Permanent bonding by oxygen plasma can be used to maximize adhesion between PDMS and glass, but it would prohibit disassembly of the chips and tissue collection.</li>
	<li>Coat the surface of the microchannel in PDMS device and microscope glass with 30 &#956;L of diluted basement membrane matrix in DMEM/F12 (1:40) by making a droplet on one side of the inlet of the channel and then aspirate the solution from the other side of the inlet with a pipette or suction pump (Figure 3A). Do not aspirate too much volume of solution to avoid bubble contamination.</li>
	<li>Then, incubate the PDMS device for 1 hour at room temperature or overnight at 4 &#176;C in a secondary container (e.g., Petri dish).</li>
</ol>
7. Motor nerve organoid (MNO) formation
<ol>
	<li>Replace the coating solution with pre-warmed 150 &#181;L of Maturation medium supplemented with 20 ng/mL of BDNF just before use.</li>
	<li>Then, place the MNS from step 5.2.9 into the inlet of the microchannel using a micropipette with a wide-bore tip. MNS can spontaneously settle down at the bottom of the device by gravity. Do not apply too much pressure when injecting the MNS (Figure 3B). 
	NOTE: If the MNS is stuck on the sidewall of a hole in a tissue culture chip, gently aspirate the solution from another side of the inlet.</li>
	<li>Fill a small reservoir (e.g., a cap of 15 mL tube) with sterile water and place it nearby the tissue culture chip in the secondary container to prevent medium evaporation. Then, place it in a 5% CO2/37 &#176;C incubator.</li>
	<li>For a medium change, aspirate the exhausted culture medium from the center of the medium reservoir (Figure 3C). Do not aspirate all the medium and the tissue.</li>
	<li>Gently add fresh Maturation medium (with BDNF). The medium should be changed every 2-3 days. Do not dry the culture medium at any time during the culture. Axons grow from an MNS into the channel and spontaneously assemble into a single bundle in 2-3 weeks, which result in the formation of an MNO. MNO can be cultured for more than additional 1 month in the device.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td>
			<td>Sigma</td>
			<td>440302</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#181;l Wide Bore Pipet Tips</td>
			<td>BMBio</td>
			<td>BMT-200WRS</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Violamo</td>
			<td>2-8588-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>Wako</td>
			<td>020-12913</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma</td>
			<td>D5942</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor reduced Matrigel (basement membrane matrix)</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol (IPA)</td>
			<td>Wako</td>
			<td>166-04836</td>
			<td></td>
		</tr>
		<tr>
			<td>Knock Out Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Sigma</td>
			<td>SML0559</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100x) (NEAA)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Glass</td>
			<td>Matsunami</td>
			<td>S9111</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stem Cell Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Wako</td>
			<td>141-08941</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime surface 96U</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-9096U</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR (passaging reagent)</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Wako</td>
			<td>186-01114</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Sigma</td>
			<td>SML1314</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Wako</td>
			<td>192-16541</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>SUMCO</td>
			<td>PW-100-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silpot 184 w/c kit</td>
			<td>Dow Toray</td>
			<td>Silpot 184 w/c kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Smi32 Antibody</td>
			<td>Biolegend</td>
			<td>801701</td>
			<td></td>
		</tr>
		<tr>
			<td>SU5402</td>
			<td>Sigma</td>
			<td>SML0443</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express liquid without phenol red (dissociation solution)</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Wako</td>
			<td>030-24021</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Osaki, T., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61544">Three-Dimensional Motor Nerve Organoid Generation.</a> J. Vis. Exp. (2020).
This video demonstrates the method of creating motor nerve organoids by first culturing human induced pluripotent stem cells in a special medium to form spheroids, then differentiating them into motor neurons using specific inhibitors, and finally cultivating them in a microfluidic chip to extend axons and form motor nerve organoids.

<img alt="Figure 1" src="/files/ftp_upload/22398/22398_Figure1.jpg" /> 
Figure 1: The dimension of PDMS tissue culture chip.
(A) Photomask of the tissue culture chip. (B) Dimensions of microchannel in the tissue culture chip. The diameter of the base chamber for holding motor neuron spheroid is 2 mm and the hole of PDMS above the chamber is 1.5 mm. The width and height of a microchannel bridging two chambe...

1. SU-8 mold fabrication by photolithography
NOTE: This procedure involves hazardous chemicals. Use fume hood and PPE throughout.

	Clean the silicon ...

Take human induced pluripotent stem cells, or iPSCs, in a nutrient-rich stem cell medium with Rho-kinase inhibitors.
The inhibitors enter the cells and block Rho-kinase enzymes, preventing apoptotic cell death.
The cells utilize the nutrients to proliferate and spontaneously aggregate to form a three-dimensional cell cluster called a spheroid.
Gradually, replace the medium with a neural induction medium. Signaling modulators from the medium drive the differentiation of stem cells into neural cells.
Replace with a growth-factor-rich neural medium to differentiate the spheroid cells into motor neurons and form a motor neuron spheroid or MNS.
Transfer these MNS to a polymer-coated microfluidic chip containing a maturation medium.
The nerve growth factors in the medium stimulate the axons or longer projections of neurons to grow from the spheroid and extend through the microchannel of the chip.
This forms a motor nerve organoid, a three-dimensional model of motor nerve tissue.

39 visualizações. Desenvolvimento de organoides nervosos motores derivados de iPSC humanos usando um chip microfluídico

Vídeo: Desenvolvimento de organoides nervosos motores derivados de iPSC humanos usando um chip microfluídico - Experimento

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

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Biologia do Desenvolvimento

Structured Motor Rehabilitation After Selective Nerve Transfers

After severe nerve injuries, selective nerve transfers provide an opportunity to restore motor and sensory function. Functional recovery depends both on the successful re-innervation of the targets in the periphery and on the motor re-learning process entailing cortical plasticity. While there is an increasing number of methods to improve rehabilitation, their routine implementation in a clinical setting remains a challenge due to their complexity and long duration. Therefore, recommendations for rehabilitation strategies are presented with the aim of guiding medical doctors and therapists through the long-lasting rehabilitation process and providing step-by-step instructions for supporting motor re-learning.
Directly after nerve transfer surgery, no motor function is present, and therapy should focus on promoting activity in the sensory-motor cortex areas of the paralyzed body part. After about two to six months (depending on the severity and modality of injury, the distance of nerve regeneration and many other factors), the first motor activity can be detected via electromyography (EMG). Within this phase of rehabilitation, multimodal feedback is used to re-learn the motor function. This is especially critical after nerve transfers, as muscle activation patterns change due to the altered neural connection. Finally, muscle strength should be sufficient to overcome gravity/resistance of antagonistic muscles and joint stiffness, and more functional tasks can be implemented in rehabilitation.

Here, we present a protocol for the motor rehabilitation of patients with severe nerve injuries and selective nerve transfer surgery. It aims at restoring the motor function proposing several stages in patient education, early-stage therapy after surgery and interventions for rehabilitation after successful re-innervation of the nerve&#8217;s target.

Reabilitação motora estruturada após transferências de nervos seletivos

After severe nerve injuries, selective nerve transfers provide an opportunity to restore motor and sensory function. Functional recovery depends both on ...

Medicina

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent development of procedures describing the generation of human cerebral organoids, derived from human induced pluripotent stem cells (iPSCs), has the potential to fundamentally transform the ability to model and understand the aging of the human brain and related pathogenic processes. Here, an optimized protocol for generating, maintaining, aging, and characterizing human iPSC-derived cerebral organoids is presented. This protocol can be implemented to generate brain organoids in a reproducible manner and serves as a step-by-step guide, incorporating the latest techniques that result in improved organoid maturation and aging in culture. Specific issues related to organoid maturation, necrosis, variability, and batch effects are being addressed. Taken together, these technological advances will allow the modeling of brain aging in organoids derived from a variety of young and aged human donors, as well as individuals afflicted with age-related brain disorders, allowing the identification of physiologic and pathogenic mechanisms of human brain aging.

The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis.

Fabricação robusta de tecidos para cultura de longo prazo de organoides cerebrais derivados de iPSC para pesquisa de envelhecimento

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent ...

Developing Human iPSC-Derived Motor Nerve Organoids Using a Microfluidic Chip

Transcrição

Developing Human iPSC-Derived Motor Nerve Organoids Using a Microfluidic Chip