developing an in vitro model of zika virus infection in cerebral organoids

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Bring Earle&#39;s 1X Balanced Salt Solution, 1X PBS, and Neural Induction medium to 37 degrees Celsius in a hot water bath. Next, dilute Zika virus of a known concentration in 1X Earle&#39;s solution to the target multiplicity of infection which is typically between 0.1 and 10. Using a P200 pipette, carefully remove all medium from each organoid well, then, quickly wash the organoids with 200 microliters of warm 1X PBS.
When removing the media from each well, it is critical that one does not touch the organoid or suck it into the pipette. This can cause irreparable damage to the organoid. Should this happen, it is best to discard the organoid.
Next, carefully remove all the remaining liquid from each organoid well using a single-channel P200 pipette. Then, quickly add 50 microliters of either 1x Earle&#39;s solution for a mock infection or Zika virus solution to each well. Ensure that each organoid is completely submerged. When finished, place the plate back into a 37 degrees Celsius incubator for two hours.
After a viral exposure is completed. Wash each well of the organoids with 200 microliters of PBS. Allow organoids to settle for 15 seconds, and then remove the PBS using a single-channel P200 pipette. Finally, add 200 microliters of fresh neural induction medium to each well, and place the plate back into the incubator.

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1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free stem cell maintenance media (SCMM) with a vitronectin or Geltrex substrate. If using an alternative, high-protein stem cell culturing media, it is advisable to transition SC cultures to SCMM for at least two passages before initiating organoid formation. The protocol has not been tested with feeder-based SC culture methods.
<ol>
	<li>Using regular SC maintenance methods, bring cultures to 50%-70% confluence. This is typically 3-4 days after passaging, though this can be dependent on culture method and cell line. 
	Note: Approximately 2 wells from a 6-well plate are needed to produce a full 96-well plate of organoids.</li>
	<li>Inspect cultures via brightfield microscopy at 10X-20X magnification to ensure healthy colony morphology, with no detectable differentiation.</li>
	<li>Bring SCMM to 37 &#176;C in a hot water bath and thaw a 50 &#181;L vial of 50 mM Y-27632 (Rock inhibitor) and 2 mL of enzymatic detachment reagent to room temperature.</li>
	<li>Prepare an ultra-low attachment (ULA) U-bottom 96 well plate, a multichannel P200 pipette, and a 25 mL reagent reservoir.</li>
	<li>Aliquot 45 mL of SCMM in a 50-mL conical tube and add 45 &#181;L of 50 mM Y-27632. Mix thoroughly by triturating.</li>
	<li>Vacuum aspirate the two wells containing SCs, and quickly add 1 mL of enzyme-free detachment reagent to each well using a P1000 pipette. Incubate the plate in a 37 &#176;C incubator for 4 min.</li>
	<li>Vacuum aspirate the treated wells and add 1 mL of enzymatic detachment reagent to each well using a P1000 pipette.
	<ol>
		<li>Incubate the plate in a 37 &#176;C incubator for an additional 5 min.</li>
		<li>Remove the plate and examine the treated wells. Gently tap on the plate to break up aggregated cells. If large cell clusters are still visible, incubate the plate for 2 additional min at 37 &#176;C. Repeat this step until clusters are no longer visible by the naked eye.</li>
	</ol>
	</li>
	<li>Once the cells are properly dissociated, add 1 mL of SCMM to each well to deactivate the dissociation enzymes. Pool the 4 mL of cell suspension in a 50-mL conical tube and take a small aliquot for cell counting.
	<ol>
		<li>Return any remaining, untreated wells in the maintenance plate to the 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>While counting, centrifuge the cell suspension at 300 x g for 5 min.</li>
	<li>Determine the total number of cells and calculate the amount of SCMM + Y-27632 solution that will be needed to achieve a 60,000 cells/mL suspension.</li>
	<li>Carefully vacuum aspirate the supernatant and add the calculated volume of SCMM + Y-27632 solution. Mix 5-10 times with a 5-mL pipet, ensuring a uniform cell suspension.</li>
	<li>Immediately transfer the cell suspension into a reagent reservoir, and pipette 150 &#181;L into each well of the ULA U-bottom 96-well plate using a multichannel P200 pipette. This produces 96 individual organoids, each initially consisting of 9,000 cells.</li>
	<li>Centrifuge the plate at 150 x g for 1 min.</li>
	<li>Place the plate into a 37 &#176;C incubator, and do not disturb the plate for 48 h.</li>
</ol>
2. Cerebral Organoid Maintenance (Day 2)
<ol>
	<li>Prepare 17 mL of SCMM with 17 &#181;L of 50 mM Y-27643 in a 50-mL conical tube. Warm the SCMM + Y-27632 solution to 37 &#176;C in a hot water bath.</li>
	<li>Take the organoid plate from the incubator and using a multichannel P200 pipette set to 75 &#181;L, slowly draw up medium from the edge of the well on the first row. Once the medium has been drawn, quickly expel the medium back into the well to lift up loose cells and organoids. Repeat this for each row. 
	Note: This technique, hereafter referred to as a &#34;dispersion,&#34; lifts cellular debris and the organoid into suspension. The organoid rapidly sinks to the bottom of the well, while the smaller debris remains in suspension, allowing it to be removed with a media change. This helps to prevent the organoid from resting in cellular debris during culture.
	<ol>
		<li>After all wells have been dispersed, wait for 15 s to ensure that the organoids have sunk to the bottom of each well.</li>
	</ol>
	</li>
	<li>Slowly and carefully aspirate 75 &#181;L of medium from each well using the multichannel P200 pipette and dispense into a waste reservoir. 
	Note: Most users occasionally aspirate an organoid during regular feeds. This can occur roughly 1% of the time. Please plan accordingly to ensure that enough organoids survive to experimental time points.</li>
	<li>Transfer SCMM + Y-27632 solution into a reagent reservoir and dispense 150 &#181;L into each organoid well using the multichannel P200 pipette.</li>
	<li>Place the plate back into a 37 &#176;C incubator.</li>
</ol>
3. Cerebral Organoid Maintenance (Day 3)
<ol>
	<li>Warm 17 mL of SCMM to 37 &#176;C, this time without Y-27643.</li>
	<li>With the multichannel P200 pipette set to 100 &#181;L, disperse all wells of the organoid plate (see step 2.2). Wait 15 s to ensure that organoids have sunk to the bottom of their wells.</li>
	<li>Prepare a waste reservoir and transfer warmed SCMM into a source reservoir.</li>
	<li>Carefully aspirate 125 &#181;L of medium from each well and replace with 150 &#181;L of fresh SCMM using the multichannel P200 pipette.</li>
	<li>Place the plate back into a 37 &#176;C incubator.</li>
</ol>
4. Cerebral Organoid Maintenance (Day 4+)
Note: Conduct this regular maintenance every other day until infection.
<ol>
	<li>Before beginning the feed on day 4, prepare Neural Induction (NI) medium.
	<ol>
		<li>Thaw a 250 &#181;L aliquot of 2 mg/mL heparin, along with one 5 mL vial of 100X N-2 Supplement. Add the 250 &#181;L of heparin, 5 mL of 100X N-2, and 5 mL of 100X Minimum Essential Medium-Non-Essential Amino Acids (MEM-NEAA) to 500 mL of Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) + GlutaMAX. Sterile filter the mixed solution in a 500 mL filter. 
		&#8203;Note: Keep NI at 4 &#176;C when not in use; NI lasts up to two weeks before fresh medium should be prepared.</li>
	</ol>
	</li>
	<li>Warm 17 mL of NI medium to 37 &#176;C.</li>
	<li>With the multichannel P200 pipette set to 100 &#181;L, disperse all wells of the organoid plate (see step 2.2). Wait 15 s to ensure that organoids have sunk to the bottom of their wells.</li>
	<li>Prepare a waste reservoir and transfer warmed NI into a source reservoir.</li>
	<li>Carefully aspirate 125 &#181;L of medium from each well and replace with 125 &#181;L of fresh NI using the multichannel P200 pipette.</li>
	<li>Place the plate back into a 37 &#176;C incubator.</li>
</ol>
5. Organoid Infection with zika virus (ZIKV) (~Day 24)
NOTE: After approximately 3 weeks of differentiation, there will be large neuroepithelial structures and rosettes visible within the organoid that will be highly susceptible to ZIKV infection.
<ol>
	<li>Bring Earle&#39;s 1X Balanced Salt Solution (EBSS), 1X phosphate-buffered saline (PBS), and NI to 37 &#176;C in a hot water bath.</li>
	<li>Dilute ZIKV of known concentration in 1X EBSS to the target infection multiplicity of infection (MOI). 
	Note: Titrations of different MOIs are recommended to achieve a viral dose response. This may vary by virus strain; for ATCC VR-1838 and ATCC VR-1843, typical MOI values range between 0.1 and 10).</li>
	<li>Carefully remove all medium from organoid well using a single channel P200 pipette. Quickly add 200 &#181;L of 1X PBS to each well using a P200 multichannel pipette to wash the organoids.</li>
	<li>Carefully remove all medium from organoid well using a single channel P200 pipette. Quickly add 50 &#181;L of 1X EBSS-only (mock infection) or ZIKV virus solution to well and ensure that the organoid is completely submerged. Repeat for each organoid that is to be infected for the study.</li>
	<li>Place the plate back into a 37 &#176;C incubator for 2 h. 
	Note: Incubating mock-infected organoids for this duration should not significantly impact their growth as compared to undisturbed organoids.</li>
	<li>Approximately 30 min before viral incubation is completed, aliquot 25 mL of NI into a 50-mL conical tube and bring to 37 &#176;C in a hot water bath.</li>
	<li>After viral exposure is completed, wash the organoids with 200 &#181;L of 1X PBS for each well, allow organoids to settle for 15 s, and then remove 1X PBS using a single channel P200 pipette.</li>
	<li>Add 200 &#181;L of fresh NI to each well, and place back into the incubator.
	<ol>
		<li>Optional: For a day 0 time point, organoids may be imaged one hour after infection.</li>
	</ol>
	</li>
	<li>Continue to culture by replacing 125 &#181;L NI every other day using the dispersion method (see step 4). Be sure to properly dispose of spent media and tips, as this contains infectious ZIKV particles.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Xeno-Free Stem Cell Maintenance Media (SCMM), TeSR-E8</td>
			<td>StemCell Technologies</td>
			<td>5940</td>
			<td>Use for stem cell maintenance and early stages of organoid formaiton. Store at 4 &#176;C for up to two weeks after prepared from individual components</td>
		</tr>
		<tr>
			<td>Enzyme-Free Detachment Reagent, ReLeSR</td>
			<td>StemCell Technologies</td>
			<td>5873</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic Detachment Reagent, Accutase</td>
			<td>Gibco</td>
			<td>A11105-01</td>
			<td>Store aliquoted at -20 &#176;C, do not keep thawed for longer than necessary</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck Chem</td>
			<td>S1049</td>
			<td>Reconstitute to 50mM in DMSO, and keep at -20 &#176;C in 50&#181;L aliquots.</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate-supplemented DMEM/F12 (1X)</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td>(+)2.438 g/L sodium bicarbonate, (+)sodium pyruvate</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (MEM-NEAA)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Adrich</td>
			<td>H4784-1G</td>
			<td>Reconstitute to 2 mg/mL in sterile DI water and store at -20 &#176;C in 250&#181;L aliquots</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (EBSS)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30029.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter (500mL)</td>
			<td>Millipore</td>
			<td>220009-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Ultra-Low Attachment Plate, 96-Well, Round Bottom</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Residual Volume Reagent Reservoir (25mL)</td>
			<td>Integra</td>
			<td>4312</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-200XLS+</td>
			<td>Rainin</td>
			<td>17013810</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Centrifuge, Culture Plate-Compatible</td>
			<td>Thermo Scientific</td>
			<td>75007210</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Plate Centrifuge Rotor</td>
			<td>Thermo Scientific</td>
			<td>75005706</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero Cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td>For virus expansion, if necessary</td>
		</tr>
		<tr>
			<td>Zika Virus (Uganda Strain)</td>
			<td>ATCC</td>
			<td>VR-1838</td>
			<td>Other strains may be used</td>
		</tr>
		<tr>
			<td>Zika Virus (Puerto Rico Strain)</td>
			<td>ATCC</td>
			<td>VR-1843</td>
			<td>Other strains may be used</td>
		</tr>
	</tbody>
</table>

Source: Salick, M. R., et al. <a href="https://app.jove.com/v/56404">Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free ...

Take a multiwell plate containing human cerebral organoids in a neural induction medium.
The organoids contain stem cells, radial glial cells, neural progenitor cells or NPCs, and neurons arranged in a radial pattern called a rosette, mimicking the developing brain.
Remove the medium and wash the organoids with a buffer.
Add Zika virus suspended in a buffer to the test wells and only buffer to the control wells.
Incubate, allowing the virus to bind to its receptors on the NPCs and become internalized.
Wash to remove non-internalized viruses, then add fresh neural induction medium and incubate.
The internalized viruses use the host cell machinery to form new viral particles.
Viral multiplication triggers a cellular stress response that induces apoptosis, reducing the number of NPCs available for organoid development.
Over time, the infected organoids exhibit reduced size and disrupted morphology, mirroring abnormal brain development in Zika virus-infected humans.

13 ビュー. 脳オルガノイドにおけるジカウイルス感染のin vitroモデルの開発

ビデオ: 脳オルガノイドにおけるジカウイルス感染のin vitroモデルの開発 - 実験

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

培養ひと胎児脳神経幹細胞の免疫細胞化学的解析のジカ ウイルス感染症

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

JoVE Journal

発生生物学

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

免疫・感染学

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

マウスの複数の臓器からのジカウイルスの単離と定量

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

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Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids