This work was supported by a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW) research fund and a grant from Start2Cure (0-TI-01). We thank Leisha Kopp for her input and advice in the setup of the protocol. Figures were created using Biorender.

All experimental procedures were approved by the institutional animal care and use committee of the Royal Netherlands Academy of Sciences (KNAW) and were in accordance with the Dutch Law on Animal Experimentation under project number AVD8010020199126. In Figure 1, a schematic overview of the complete protocol is provided. From seeding cells to AAV purification, the protocol takes 6 days to complete.
1. Reagent preparation
<ol>
	<li>Plasmid purification
	<ol>
		<li>Perform plasmid extraction as described by the manufacturer with a few adjustments to ensure sterility of the plasmids.</li>
		<li>Prepare 70% ethanol using sterile distilled water and absolute ethanol.</li>
		<li>After the isopropanol centrifugation step, dry plasmids in the flow cabinet hood and add sterile 70% ethanol to the tubes. After ethanol precipitation, dry the plasmids in the flow cabinet hood and resuspend the plasmid in sterile distilled water. Take care to use sterilized microcentrifuge tubes.</li>
		<li>Take an aliquot for quantification, restriction analysis, and, if needed, sequencing. It is advised to check the integrity of the inverted terminal repeats (ITRs) as mutations can have a negative impact on yield. Adjust the sample concentration to 1 &#181;g/&#181;L.</li>
	</ol>
	</li>
	<li>Preparation of 10x Tween lysis buffer
	<ol>
		<li>Dissolve 30.3 g Tris buffer and 2.033 g MgCl2.6H2O in 400 mL of sterile distilled water, set pH to 8.0, and increase total volume to 450 mL.</li>
		<li>Keep Tween 20 sterile, add 50 mL of Tween 20 to buffer solution in the hood, and filter sterilize using a 0.2&#160;&#181;M vacuum filter.</li>
	</ol>
	</li>
	<li>Iodixanol dilutions
	<ol>
		<li>Iodixanol solution is provided at 60% concentration. Use the starting solution to make 15%, 25%, and 40% solutions.</li>
		<li>For 15% solution, dilute 60 mL of 60% solution with 48 mL of 5 M NaCl and 48 mL of PBS-MK (5x PBS with 5 mM MgCl2 and 12.5 mM KCl), and add distilled water up to 240 mL.</li>
		<li>For 25% solution, dilute 67 mL of 60% solution and 32 mL of PBS-MK. Add distilled water up to 160 mL. Mix well and add 1.6 mL of phenol red solution.</li>
		<li>For 40% solution, dilute 160 mL of 60% solution with 48 mL of PBS-MK and add distilled water up to 240 mL. For 60% solution, add 1 mL of phenol red to 100 mL of 60% solution.</li>
	</ol>
	</li>
	<li>PBS 5% sucrose solution
	<ol>
		<li>Add 25 g of sucrose to a 500 mL bottle of DPBS without calcium or magnesium. Shake well and filter using a 0.2 &#181;M bottle vacuum filter.</li>
	</ol>
	</li>
	<li>Polyethylene glycol (PEG) 8000 solution
	<ol>
		<li>Dissolve 400 g of PEG 8000 and 24 g of NaCl into sterile distilled water and adjust to a final volume of 1000 mL. Stir with heating until fully dissolved. Adjust pH to 7.4 using pH paper.</li>
		<li>Filter sterilize using a 0.2&#160;&#181;M bottle vacuum filter to obtain a 40% PEG 8000 solution. Note that filtration will take a while due to the viscosity of the solution-store at 4 &#730;C.</li>
	</ol>
	</li>
</ol>
2. Culture of HEK293 suspension cells
<ol>
	<li>Use wipes soaked in 70% ethanol for cleaning. Clean biosafety hood, prepare and clean all materials needed for culturing cells.</li>
	<li>Prewarm 30 mL of suspension cell medium to 37 &#176;C in a 50 mL conical culturing tube. Retrieve viral production cells from liquid nitrogen. Quickly thaw cells in a 37 &#176;C water bath. Just before the vial is completely thawed, clean it with a wipe soaked in 70% ethanol and transfer the vial to the biosafety hood. 
	NOTE: The details of the viral production cells used here are provided in the Table of Materials.</li>
	<li>Quickly transfer cells to the prewarmed 50 mL conical culturing tube. Culture cells in a shaking incubator at 37 &#176;C, 80% humidity, 8% CO2,&#160;200 revolutions per min (RPM), and a shaking diameter of 50 mm. Passage cells, once viability is above 90% and cell density is above 1-3 x 106 cells/mL. 
	NOTE: The incubator used has a shaking diameter of 50 mm; culture conditions should be adjusted for a different shaking diameter.</li>
	<li>Passage cells 3-4 days after thawing. Check culture tube(s) to make sure there are no signs of contamination; for example, fungus will appear as a discolored (black or green) ring.</li>
	<li>Prepare 400 &#181;L of 0.4% trypan blue solution per sample using a 1 mL stripette.</li>
	<li>Quickly retrieve suspension cells from the incubator. Immediately extract 500 &#181;L from the tube using a 1 mL stripette. Gently pipet up and down.</li>
	<li>Add 100 &#181;L of cell suspension to the prepared trypan blue tube. Gently invert the tube to mix; do not pipet to mix, as this will lead to cell death. Return cells to incubator.</li>
	<li>Take 50 &#181;L of trypan blue-treated cell suspension and apply it to the hemocytometer slide. Count total viable cells and calculate the amount of cell suspension and medium needed for passage or transfection the following day.</li>
	<li>Warm medium in the incubator for 10-15 min. Add cell suspension to warmed medium and return to incubator. For passaging cells for 3 days (i.e., Monday-Thursday), set to 0.5 x 106 cells/mL; for passaging cells for 4 days (i.e., Thursday-Monday), set to 0.3 x 106 cells/mL. 
	NOTE: According to the manufacturer, viral production cells double every 26 h and should be between 3.5 x 106 and 5.5 x 106 before passaging. Cells can be kept for up to passage 20.</li>
</ol>
3. Transfection
<ol>
	<li>Day 1
	<ol>
		<li>At 24 h before transfection, culture 1 x 106 cells per/mL in 300 mL of media.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Warm medium, plasmids, and transfection reagent to room temperature. For the amounts to prepare, see Table 1.</li>
		<li>Briefly vortex plasmids and reagents. Add plasmids to the prepared medium, vortex. Add transfection reagent and vortex briefly. Do not agitate the mixture after this step. Incubate at room temperature for 30 min.</li>
		<li>In the meantime, count cells prepared on day 1. Cells should be between 2 and 2.5 x 106 cells per mL and &#62; 95% viable.</li>
		<li>After incubation, dropwise, add the transfection mixture to cells while gently swirling the tube. Incubate for 72-96 h.</li>
	</ol>
	</li>
</ol>
4. Harvesting the cells
<ol>
	<li>Day 5
	<ol>
		<li>Add 33 mL of 10x cell lysis buffer (500 mM Tris pH 8, 10% Tween 20, 20 mM MgCl2), mix by gently shaking, and incubate at 37 &#176;C for 1.5 h with shaking.</li>
		<li>Centrifuge at 3428 x g at 4 &#176;C for 60 min. Filter through a 0.45 &#181;M PES vacuum filter to clarify the cell lysate, leaving the clarified lysate in the container of the filter. 
		NOTE: Optional after filtration: take 50 &#181;L of sample for quantitative-PCR (Q-PCR).</li>
		<li>Add 90 mL of 40% PEG 8000 solution to 360 mL of filtered cell lysate.</li>
		<li>Clean the stir bar with 70% ethanol and add to the sample. Stir on ice or in the cold room at 300 RPM for 1 h. Incubate at 4 &#176;C without stirring overnight. 
		NOTE: Incubation times of 1 h to 72 h have been tested. Longer incubation times have a negative effect on overall protein precipitation.</li>
	</ol>
	</li>
</ol>
5. Iodixanol purification
<ol>
	<li>Day 6
	<ol>
		<li>Transfer the entire PEG precipitated culture volume sample to a clean, large conical tube and centrifuge at 2820 x g and&#160;4&#176;C for 15 min.</li>
		<li>Discard the supernatant and resuspend the PEG pellet in 15 mL of DPBS with calcium and magnesium. The pellet is difficult to resuspend; take the time to do this carefully.</li>
		<li>Transfer the resuspended part to a clean 50 mL tube. PEG will remain on the sides of the bioreactor tube. Add an extra 10 mL, taking care to gather all the PEG precipitate. Transfer all to a 50 mL container for a total volume of 25-30 mL.</li>
		<li>Add 40 &#181;L of DNaseI (10 U/&#181;L). Place in the incubator for 1 h 37&#176;C. 
		NOTE: Optional after incubation of DNase: take 50 &#181;L of sample for Q-PCR.</li>
		<li>Clean stir bars that were used for PEG precipitation well with chloride solution followed by 70% ethanol. Leave stir bars in 70% ethanol for the next experiment.</li>
		<li>Fill a 25 mm x 89 mm polyallomer tube using a glass Pasteur pipette with 15.5 mL of concentrated cell lysate in each tube. After adding cell lysate, exchange the glass Pasteur pipette for a new one.</li>
		<li>Add iodixanol solutions from 15%-60%: infuse 9 mL of the 15% iodixanol solution gently beneath the cell lysate. Next, add 5 mL of the 25% and 40% iodixanol solutions, and lastly, add 5 mL of the 60% iodixanol solution.</li>
		<li>Top off the tube using a syringe with DPBS +/+ to remove most of the air bubbles, taking care not to disturb iodixanol layers. Seal the tube using an electrical tube topper.</li>
		<li>Centrifuge in a non-swing rotor for 1 h 10 min at 490,000 x g at 16 &#176;C in an ultracentrifuge. Remove from ultracentrifuge, assemble the tubes in a metal clasp, and prepare collection tubes. Open the rotor in the hood in case of spills during spinning.</li>
		<li>Prepare one 50 mL tube (for discarding the rotor tube), one 15 mL tube (for collecting virus), a 5 mL syringe, and a 30G and 19G needle per gradient.</li>
		<li>Puncture a hole in the top of the tube with a 30G needle. Leave the needle in place. Place a 19G needle on the syringe.</li>
		<li>Carefully puncture the tube just below the 40%/60% interface, which can be seen by the phenol red indicator. Make sure the needle bevel is facing the 40% layer.</li>
		<li>Remove the 30G needle from the top of the tube using the non-dominant hand. With the needle beveled up, slowly extract the virus/iodixanol. The aim is to extract between 3 mL and 4.5 mL. About halfway and well into the 40%/clear layer, rotate the needle for the bevel to face down and continue to extract to avoid collection from the protein layer.</li>
		<li>Prepare the 50 mL tube with the non-dominant hand, carefully extract the needle while placing the tube in the 50 mL tube and discard. Dilute the AAV/iodixanol suspension 5x in DPBS by filling up to 15 mL.</li>
		<li>Transfer to a centrifugal filter tube and centrifuge at 3428 x g, 4 &#176;C for 10 min. Discard flow-through; gently remove the holder of the filter and tip the bottom container into a waste bottle. Add 15 mL of PBS-5% sucrose to filtrate and resuspend.</li>
		<li>Centrifuge at 3428 x g at 4 &#730;C for 20 min. Repeat buffer exchange at least 3x. Store virus (~150-250 &#181;L) at 4 &#176;C (PHP.B variants a maximum of 3 months) or aliquot into 50 &#181;L aliquots and store at -80 &#176;C for long term. 
		NOTE: PHP.B capsid variants are sensitive to freeze/thaw, so this should be avoided.</li>
		<li>Perform titration as described in a previous protocol10.</li>
	</ol>
	</li>
</ol>

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

<ol>
	<li>Zhou, K., Han, J., Wang, Y., Zhang, Y., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routes+of+administration+for+adeno-associated+viruses+carrying+gene+therapies+for+brain+diseases.">Routes of administration for adeno-associated viruses carrying gene therapies for brain diseases.</a> Front Mol Neurosci. 15, 988914 (2022).</li><li>Pietersz, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhP.B+Enhanced+adeno-associated+virus+mediated-expression+following+systemic+delivery+or+direct+brain+administration.">PhP.B Enhanced adeno-associated virus mediated-expression following systemic delivery or direct brain administration.</a> Front Bioeng Biotechnol. 9, 679483 (2021).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Several+rAAV+vectors+efficiently+cross+the+blood&#8211;brain+barrier+and+transduce+neurons+and+astrocytes+in+the+neonatal+mouse+central+nervous+system.">Several rAAV vectors efficiently cross the blood&#8211;brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system.</a> MolTher. 19 (8), 1440-1448 (2011).</li><li>Deverman, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-dependent+selection+yields+AAV+variants+for+widespread+gene+transfer+to+the+adult+brain.">Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.</a> Nat Biotechnol. 34 (2), 204-209 (2016).</li><li>Chan, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+AAVs+for+efficient+noninvasive+gene+delivery+to+the+central+and+peripheral+nervous+systems.">Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.</a> Nat Neurosci. 20, 1172-1179 (2017).</li><li>Goertsen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+capsid+variants+with+brain-wide+transgene+expression+and+decreased+liver+targeting+after+intravenous+delivery+in+mouse+and+marmoset.">AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.</a> Nat. Neurosci. 25 (1), 106-115 (2022).</li><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat Biotechnol. 27 (1), 59-65 (2008).</li><li>Challis, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+AAV+vectors+for+widespread+and+targeted+gene+delivery+in+rodents.">Systemic AAV vectors for widespread and targeted gene delivery in rodents.</a> Nat Protoc. 14 (2), 379-414 (2019).</li><li>Fripont, S., Marneffe, C., Marino, M., Rincon, M. Y., Production Holt, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=purification,+and+quality+control+for+adeno-associated+virus-based+vectors.">purification, and quality control for adeno-associated virus-based vectors.</a> J Vis Exp. (143), e58960 (2019).</li><li>Fagoe, N. D., Eggers, R., Verhaagen, J., Mason, M. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+compact+dual+promoter+adeno-associated+viral+vector+for+efficient+delivery+of+two+genes+to+dorsal+root+ganglion+neurons.">A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons.</a> Gene Thr. 21 (3), 242-252 (2014).</li><li>Verhaagen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+gene+therapy,+methods+and+protocols.">Retinal gene therapy, methods and protocols.</a> Meth Mol Biol. 1715, 3-17 (2018).</li><li>Nasse, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addgene+AAV+data+hub:+A+platform+for+sharing+AAV+experimental+data.">Addgene AAV data hub: A platform for sharing AAV experimental data.</a> Nat Meth. 20 (9), 1271-1272 (2023).</li><li>Grieger, J. C., Soltys, S. M., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+adeno-associated+virus+vectors+using+suspension+HEK293+cells+and+continuous+harvest+of+vector+from+the+culture+media+for+GMP+FIX+and+FLT1+clinical+vector.">Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector.</a> Mol Ther. 24 (2), 287-297 (2016).</li><li>Blessing, D., D&#233;glon, N., Schneider, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+mammalian+cells,+methods+and+protocols.">Recombinant protein expression in mammalian cells, methods and protocols.</a> Meth Mol Biol. 1850, 259-274 (2018).</li></ol>

A free sample of TransIT was provided by Mirus for initial testing, after which the reagent was purchased for further use. The authors have no other competing financial interests to report.

Systemic administration of AAV is a powerful tool for&#160;gene transfer to the CNS; however, the production of AAV is an expensive and laborious process. By using suspension cells, labor and plastics are reduced compared to the adherent culture of HEK293T on 15 cm2 plates. Furthermore, the conical tubes implemented here are easy to handle and maximize the use of laboratory space. The protocol was set up by two researchers and subsequently applied by others in the lab. A series of productions by three independ...

Adeno-associated virus (AAV) was discovered in 1965 and has since been used in a myriad of applications1. AAVs have been applied in neuroscience research to study gene and neuronal function, map neurocircuits, or produce animal models for disease2. Traditionally, this is done by injecting directly at the site of interest, as most natural serotypes do not cross the blood-brain barrier or need a high dose to do so1,2,3.
With the discovery of AAV.PHP.B4 and next-generation capsids such as AAV.PHP.eB5 and AAV.CAP-B106, it is possible to target the central nervous system (CNS) using a simple systemic injection. Spatial mapping reveals the cells targeted by AAV.PHP.eB at cellular level6,7. In combination with specific promoters/enhancers, these capsids offer extensive opportunities for neuroscientists to study genes and brain function by non-invasive AAV delivery4,8.
While a lower dose is needed for AAV.PHP.eB (typically 1 to 5 x 1011 Genome Copies (GC)/mouse) compared to AAV9 (4 x 1012 GC/mouse)7, still more vector needs to be produced compared to direct injection strategies (typically 1 x 109 GC/&#181;L injection). Most natural serotypes can be produced using the classical adherent cell culture system in combination with iodixanol purification9,10,11,12. For AAV.PHP.eB this entails a labor-intensive process to culture and transfect cells to obtain sufficient vectors for one experiment8. Therefore, the production of AAV in suspension cell culture in conical tubes was developed. Conical tubes, with a capacity of up to 300 mL, are compact, saving both incubator space and plastics. Suspension cells are much easier to culture and handle in large amounts than adherent cells on 15 cm plates. The transfection components of the protocol remain the same. Therefore, plasmids previously used with the adherent system can easily be used in this protocol based on production in suspension cells. The protocol was successfully transferred to other researchers in the laboratory and successfully used for various capsids and constructs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>39 mL, Quick-Seal Round-Top Polypropylene Tube, 25 x 89 mm - 50Pk</td>
			<td>Beckman Coulter</td>
			<td>342414</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter 600 mL conical tubes, for rotor S-4x1000,&#160;</td>
			<td>eppendorf</td>
			<td>5920701002</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter Plate fits 16 bioreactors of 600 ml</td>
			<td>Infors HT/ TPP</td>
			<td>587633</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol-tight caps, for 750 mL round buckets</td>
			<td>eppendorf</td>
			<td>5820747005</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5920 R G, 230 V, 50-60 Hz, incl. rotor S-4x1000, round buckets and adapter 15 mL/50 mL conical tubes</td>
			<td>eppendorf</td>
			<td>5948000315</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Gibco</td>
			<td>15230147</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I recombinant, RNase-free</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I recombinant, RNase-free</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Gibco</td>
			<td>14040091</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Disposable PES Filter Units 0,2</td>
			<td>Fisher</td>
			<td>FB12566504</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Disposable PES Filter Units 0,45</td>
			<td>Fisher</td>
			<td>FB12566505</td>
			<td></td>
		</tr>
		<tr>
			<td>Holder for 50 ml culture tubes also fits falcon tube</td>
			<td>Infors HT/ TPP</td>
			<td>31362</td>
			<td></td>
		</tr>
		<tr>
			<td>Holder for 600 ml cell culture tube</td>
			<td>Infors HT/ TPP</td>
			<td>66129</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Minitron 50 mm</td>
			<td>Infors HT</td>
			<td>500043</td>
			<td></td>
		</tr>
		<tr>
			<td>LV-MAX Production Medium</td>
			<td>Gibco</td>
			<td>A3583401</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Tray Universal</td>
			<td>Infors HT/ TPP</td>
			<td>31321</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep - Iodixanol</td>
			<td>Serumwerk bernburg</td>
			<td>1893</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)</td>
			<td>Poly-sciences</td>
			<td>24765-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>72420100</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>TransIT-VirusGEN</td>
			<td>Mirus</td>
			<td>Mir 6706</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>5250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TubeSpin Bioreactors-50ml</td>
			<td>TTP</td>
			<td>87050</td>
			<td></td>
		</tr>
		<tr>
			<td>TubeSpin Bioreactors-600ml</td>
			<td>TTP</td>
			<td>87600</td>
			<td></td>
		</tr>
		<tr>
			<td>Viral Production Cells</td>
			<td>Gibco</td>
			<td>A35347</td>
			<td></td>
		</tr>
		<tr>
			<td>Vivaspin 20 MWCO 100 000</td>
			<td>Cytvia</td>
			<td>28932363</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of high-yield adeno associated vector batches using hek293 suspension cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Most academic labs use adherent HEK293T cells for AAV production8,9. While this works relatively well when small amounts of AAV are needed for direct injection, a 100-fold higher titer (minimally 1 x 1011 GC/mouse) is needed to achieve similar transduction with systemic capsids such as AAV.PHP.eB.
In this protocol, the production of AAV using suspension HEK293 cells cultured in conical tubes was established. Small-scale cult...

Read this Scientific Article Protocol about Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation at JoVE.com

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

production-high-yield-adeno-associated-vector-batches-using-hek293

Research

JoVE Journal

Neuroscience

1.9K Views.  Royal Netherlands Academy of Arts and Sciences (KNAW). Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors. 

Video: Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...