We would like to thank Dr. Robert M. Kotin (National Heart, Blood and Lung Institute, NIH) for generously providing us the AAV plasmids and Danielle Steele and Rebecca Ernst (Cincinnati Children&#39;s Hospital) for their technical assistance. This work is supported by the Start-Up fund from Cincinnati Children&#39;s Research Foundation to M.N.

See Figure 1 for an illustration summarizing the protocol.
1. Generation of baculovirus-infected TIPS cells
<ol>
	<li>Thaw one vial of Sf9 cells and immediately seed them in 50 mL of insect cell culture medium. Grow Sf9 cells in an orbital shaker incubator at 125 rpm and 28 &#176;C in round bottom flasks with a loosely attached cap for exchange of air8,9. Propagate the cells in a 1 L flask containing 200 mL of medium to obtain enough cells for TIPS cells production.</li>
	<li>Add 50 mL of Sf9 cells at 2 x 106 cells/mL in two flasks: infect one flask of Sf9 cells with 0.5 mL of baculovirus (BV)-AAV2-GFP (or therapeutic gene) and the other flask of cells with 0.5 mL of BV-AAV2-rep-cap. 
	NOTE: AAV vector plasmids were generously provided by Dr. Robert M. Kotin (NIH). Baculovirus production from bacmid DNA (Bac-to-Bac System) has been described previously8,9. The baculovirus stability during the passage is a critical problem for the scale-up production of rAAV. It is better to use a lower passage number of baculovirus (up to passage number 4). Determine the optimal volume of the baculovirus supernatant empirically by checking the baculovirus infection efficiency using flow cytometry (Figure 2) during TIPS cell production.</li>
	<li>Monitor the baculovirus infection 3-4 days post-infection by staining baculovirus gp64 in infected Sf9 cells as follows.
	<ol>
		<li>Stain 2 x 105 Sf9 cells (both uninfected and baculovirus-infected cells) with 0.5 &#181;g of mouse anti-baculovirus gp64 antibody (1:200) containing a fluorescent dye in 100 &#181;L of blocking buffer for 30 min at ambient temperature.</li>
		<li>Wash the cells with 1 mL of PBS to remove the antibody and resuspend the stained cells in 300 &#181;L of PBS containing 0.5% bovine serum albumin.</li>
		<li>Analyze the baculovirus gp64 expression on cells using flow cytometry (Figure 2). 
		NOTE: Determine the gating strategy for flow cytometry acquisition and analysis empirically. A total of 10,000 single live cells is acquired. Baculovirus gp64 expression on Sf9 cell surfaces indicates baculovirus infection. After 3-4 days, most of the Sf9 cells become infected with the baculovirus, as evidenced by baculovirus gp64 expression (Figure 2).</li>
	</ol>
	</li>
	<li>When the cells are 80%-90% viable with an increase in the diameter (Figure 3), harvest the cells and cryopreserve 1 x 107 cells in 1 mL of insect culture medium supplemented with 10% fetal bovine serum and 10% dimethyl sulfoxide in a slow-freezing container at -80 &#176;C. The next day, transfer the tube containing TIPS cells to a liquid nitrogen freezer for long-term storage. 
	&#8203;NOTE: Perform cell culture in a biosafety cabinet following the standard aseptic laboratory technique at Biosafety Level 2 (BSL-2).</li>
</ol>
2. Production of AAV vector
<ol>
	<li>Day 1: Co-culture Sf9 cells with the baculovirus-infected TIPS cells
	<ol>
		<li>Culture and grow naive Sf9 cells at 1 x 106 cells/mL at 28 &#176;C in several 2 L flasks containing 400 mL of insect cell culture medium in a shaker incubator that is needed for AAV production. After 3-4 days, the cell density is increased up to 5 x 106-6 x 106 cells/mL. 
		NOTE: CO2 and humidity are not important factors for the growth of Sf9 cells.</li>
		<li>Seed the Sf9 cells either in shake flasks or in a bioreactor as follows.
		<ol>
			<li>AAV production in flasks in an orbital shaker incubator: Use a pipette or a peristaltic pump to seed 400 mL of Sf9 culture at 2 x 106 cells/mL into a 2 L flask aseptically. Set up the orbital shaker at 125 rpm and 28 &#176;C. 
			&#8203;NOTE Use a peristaltic pump or standard pipette depending on the scale of the AAV production.</li>
			<li>AAV production in a bioreactor: Use a peristaltic pump to seed 1 L of Sf9 culture at 2 x 106 cells/mL into a 10 L bioreactor bag aseptically. Load the bag onto the Bioreactor unit for culturing at 28 &#176;C. Add 40% oxygen to the bioreactor bag at 0.1 psi. Set up the bioreactor unit at a 20&#176; angle and shake the bag at 25 rpm.</li>
		</ol>
		</li>
		<li>Thaw one vial of each BV-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap TIPS cells. Dilute the cells with 20 mL of insect culture medium and perform a viability count of the cells using trypan blue. Inoculate both TIPS cells at a ratio of 1:10,000 relative to the naive Sf9 cells cultured in a shaker flask or bioreactor. 
		&#8203;NOTE: The survival of the TIPS cells is reduced due to cryopreservation, and the recovery rate is approximately 50% when cell viability is determined with the trypan blue staining. Perform a pilot experiment to empirically determine the ratio of TIPS cells and na&#239;ve Sf9 cells before large-scale rAAV production.</li>
	</ol>
	</li>
	<li>Day 2: Culture monitoring
	<ol>
		<li>Harvest 1 mL of Sf9 culture aseptically. Stain with the Trypan blue dye to analyze the cell count, viability, and morphology.</li>
	</ol>
	</li>
	<li>Day 3: Culture monitoring and feeding
	<ol>
		<li>Harvest 1 mL of Sf9 culture and analyze the cell count, viability, and morphology. Check the status of baculovirus infection after staining the Sf9 cells as mentioned in step 1.3. 
		NOTE: Increase in cell diameter and cytopathic effect are indications of baculovirus infection. The increase in diameter precedes a decrease in cell viability, and these parameters are used to determine the cell harvest time during AAV production. Determine the optimal time point empirically.</li>
		<li>Feed the Sf9 culture with fresh insect culture medium at a ratio of 1:5 aseptically.</li>
	</ol>
	</li>
	<li>Day 4: Culture monitoring
	<ol>
		<li>Harvest 1 mL of Sf9 culture and analyze the cell count, viability, and morphology. Disconnect oxygen supply from the bioreactor bag.</li>
	</ol>
	</li>
	<li>Day 5: Culture monitoring and harvest
	<ol>
		<li>Harvest 1 mL of Sf9 culture and analyze the cell count, viability, and morphology. Harvest the culture medium and cells when Sf9 cell viability has decreased to around 50% (Figure 4).</li>
		<li>Spin the cell suspension at 2100 x g for 15 min at 4 &#176;C. Collect the supernatant and the cell pellet, and store them at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Lysis of cells and release of AAV
<ol>
	<li>Thaw the AAV producer cell pellet. Add 100 mL of cell lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 0.5 % Triton-X-100) to the cell pellet, mix vigorously, and incubate for 30 min at ambient temperature to release the AAV particles.</li>
	<li>Centrifuge at 2100 &#215; g for 30 min at 4 &#176;C and transfer the cell lysate to a new container. Mix the cell lysate and cell culture supernatant after thawing.</li>
	<li>Add 20 U/mL nuclease and 10 mM MgCl2 to the cell lysate to digest the DNA and RNA. Incubate for 2-4 h at 37 &#176;C.</li>
	<li>Filter the lysate through a 0.8 &#181;m and 0.2 &#181;mpolyethersulfone dual filtration system using a pump.</li>
	<li>Store the clarified cell lysate at 4 &#176;C overnight or purify the AAV immediately.</li>
</ol>
4. Purification of AAV vector using affinity column chromatography system
<ol>
	<li>Prepare the chromatography instrument by sequentially washing the sample and buffer lines with sterile water, 1 N sodium hydroxide, water, and phosphate-buffered saline (PBS, pH 7.4) at a flow rate of 50 mL/min.</li>
	<li>Mount the AVB Sepharose column (10.0 mL) into the chromatography system, and run 100 mL of PBS at a flow rate of 5 mL/min. 
	NOTE: Use a lower or higher volume of AVB&#160;Sepharose column depending on the scale of AAV production and set up the flow rate as suggested by the column or resin manufacturer (see Table of Materials).</li>
	<li>To collect the fractions of column pass-through solution, wash buffer, and elution buffer containing AAV particles, insert tubes into the fraction collector slots of the chromatography instrument.</li>
	<li>Equilibrate the column with PBS at a flow rate of 5.0 mL/min.</li>
	<li>Load the filtered cell lysate containing AAV particles onto the&#160;column using the chromatography machine equipped with a sample pump. Run the sample at a flow rate of 3.0 mL per min.</li>
	<li>Run PBS through the AVB&#160;Sepharose column at a flow rate of 3.0 mL/min until the ultraviolet (UV) absorbance curve (280 nm) returns to the baseline and becomes stable.</li>
	<li>Elute the AAV particles from the column with 50 mM sodium citrate (pH 3.0) buffer at a flow rate of 3.0 mL/min (Figure 5). 
	NOTE: While the AAV particles dissociate from the resin and pass through the UV detector, an elution peak of protein can be seen on the chromatogram.</li>
	<li>Immediately dilute the AAV supernatant with a one-fifth volume of 500 mM Tris-HCl (pH 8.0) to increase the pH of the AAV containing supernatant to approximately pH 5.5 to prevent pH-mediated degradation with acidic elution buffer. Further, dilute the AAV supernatant 10-fold with PBS to fully neutralize the pH. 
	NOTE: The pH value is approximately 5.5 after neutralizing the rAAV solution. After neutralizing AAV, dilute the rAAV solution 10-fold with PBS (pH 7.4) so that AAV is in a physiologic buffer.</li>
	<li>Store 1 mL of each column run-through samples, wash buffer, and 100 &#181;L of the eluted AAV supernatant to evaluate the presence of AAV by infection of the target cells.</li>
	<li>Clean the AVB&#160;Sepharose column with 100 mL of 100 mM citric acid (pH 2.1) and 100 mL of PBS (pH7.4) at a flow rate of 5.0 mL/min. Rinse the column with 20% ethanol at a flow rate of 5.0 mL/min and store at 4 &#176;C.</li>
	<li>Clean the pipelines of the chromatography instrument with the column offline position as described in section 4.1. Finally, rinse the chromatography system with 200 mL of 20% Ethanol at a flow rate of 50.0 mL/min and store in 20% ethanol.</li>
</ol>
5. Concentration and diafiltration of AAV vector using tangential flow filtration (TFF)
<ol>
	<li>Set up the TFF system equipped with a polysulfone membrane cartridge (100 kDa Molecular Weight Cut Off) to concentrate the AAV.</li>
	<li>Equilibrate TFF module with 200 mL of PBS for 10 min.</li>
	<li>Load the AAV sample&#160;by controlling the pump&#39;s flow until the sample is reduced to the desired volume while maintaining a trans-membrane pressure at 2-3 psi.</li>
	<li>Diafiltrate the retentate with 100 mL of PBS, as used in this study, or empirically define&#160;alternate buffer that supports long-term stability of AAV.</li>
	<li>After concentrating the AAV sample to the desired volume, collect the AAV sample, filter it through a 0.2 &#181;m polyethersulfonefilter, aliquot, and then store it at -80 &#176;C.</li>
</ol>
6. Infection of AAV samples into the target cells to evaluate the presence of AAV in the purification steps
<ol>
	<li>Inoculate 7 x 104 HT1080 cells/well in a 24-well plate in 500 &#181;L of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 1% L-glutamine, 1% sodium pyruvate, and 10% fetal bovine serum (FBS) the day before infection. Culture the cells in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Count the cells before infection. Add the diluted unpurified AAV bulk sample before the column run, column run-through samples, wash buffer, and eluted purified AAV sample. 
	NOTE: Count the Trypan blue-stained cells with a hemocytometer. Determine the dilution factor and volume (&#181;L) of the AAV samples empirically. The higher the AAV titers, the more dilution is needed. Ensure that the baculovirus particles of the unpurified bulk sample before the column run, column run-through samples, wash buffer are heat-inactivated at 50 &#176;C for 50 min before infecting the target cells to determine infectious titers of AAV.</li>
	<li>Two days after infection, analyze the protein expression (e.g., GFP or therapeutic gene) in the cells using a flow cytometer.</li>
	<li>Calculate infectious titers of AAV using the number of cells at the time of infection, dilution factor, and percentage of GFP+ cells with the formula: (Total number of cells x Percentages GFP+ cells x Dilution Factor) / Volume of AAV in milliliters. 
	NOTE:&#160;For example, the number of cells is 1 x 105, the percentage of GFP+ cells is 3.4%, the dilution factor is 100, and the volume of AAV added is 2 &#181;L. The titer of AAV is 1.7 x 108 infectious unit (IU)/mL. The total amount of purified AAV particles in 25 mL of the eluted fraction is 4.3 x 109 infectious units (IU)/mL (Figure 6). The total number of initial unpurified AAV particles is 1.09 x 1010 IU/mL, and purified AAV particles are 4.3 x 109 IU/mL. Therefore, the percentage of recovery is 39.4%. The percentage of GFP+ cells should be between 1%-30%, which corresponds to a linear range when used to calculate the infectious titer of AAV. If more concentrated AAV vector particles are infected into the target cells; there is a possibility that multiple copies of the AAV particles may infect a single cell that will show as a single copy of the vector. Therefore, dilute the AAV vector supernatant to obtain 1%-30% vector infection in the target cells. If the AAV vector does not have a reporter gene, stain the transgene or the therapeutic gene expressed by the AAV vector with an antibody containing a fluorescent dye that can be detected and quantitated using a flow cytometer. Not all AAV serotypes can efficiently infect HT1080 cells. Therefore, to perform the infectivity assay for other AAV serotypes, use different cell lines. Titer the AAV2-GFP particles before purification and after purification on HT1080 cells and measure the GFP expression by flow cytometry. Calculate the recovery (%) by the ratio of the number of purified AAV particles and the number of AAV particles prior to purification multiplied by 100.</li>
</ol>

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The authors declare no conflicts of interest.

The parameters used in this protocol for the process development of production, purification, and concentration of AAV vectors can be applied to both small and large-scale manufacturing of AAV vectors for gene therapy applications. The entire upstream and downstream process can be performed in a closed system compatible with the current Good Manufacturing Practices (cGMP). The major advantages of the Sf9-baculovirus system are scalability for large-scale GMP-grade AAV production at an affordable cost. The system does not...

Adeno-associated viruses (AAV) are non-enveloped human parvoviruses containing a single-stranded DNA of 4.6 kb. AAV vectors have several advantages over other viral vectors for gene therapy applications1,2,3,4. AAVs are naturally replication-incompetent, thereby, require a helper virus and host machinery for replication. AAVs do not cause any disease and have low immunogenicity in the infected host3,5. AAV can infect both quiescent and actively dividing cells and may persist as episome without integrating into the genome of the host cells (AAV rarely integrate into the host genome)1,3. These features have made AAV a desirable tool for gene therapy applications.
To generate an AAV gene transfer vector, the transgene cassette, including the therapeutic gene, is cloned between two internal terminal repeats (ITRs), which are typically derived from the AAV serotype 2. The maximum size from 5&#39; ITR to 3&#39; ITR, including the transgene sequence, is 4.6 kb6. Different capsids may have a different cell or tissue tropism. Therefore, capsids should be chosen based on the tissue or cell type intended to be targeted with the AAV vector7.
Recombinant AAV vectors are commonly produced in mammalian cell lines such as human embryonic kidney cells, HEK293 by transient transfection of the AAV gene transfer vector, AAV rep-cap, and helper virus plasmids2,3. However, there are several limitations for large-scale AAV production by transient transfection of adherent HEK293 cells. First, a large number of cell stacks or roller bottles are needed. Second, high-quality plasmid DNA and transfection reagents are needed, which increases the cost of manufacturing. Finally, when using adherent HEK293 cells, the serum is frequently needed for optimal production, complicating downstream processing1,2,3. An alternative method of AAV manufacturing involves using the insect cell line, Spodoptera frugiperda (Sf9) cells, and an insect virus called recombinant Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV or baculovirus)8,9,10. Sf9 cells are grown in serum-free suspension culture that is easy to scale up and is compatible with current good manufacturing practice (cGMP) production at a large scale, which does not require plasmid or transfection reagents. Moreover, the cost of the AAV production using the Sf9-baculovirus system is lower than the cost of using transient transfection of plasmids into HEK293 cells11.
The original rAAV production system using baculovirus-Sf9 cells used three baculoviruses: one baculovirus containing gene transfer cassette, the second baculovirus containing rep gene, and the third baculovirus containing serotype-specific capsid gene12,13. However, the baculovirus containing rep construct was genetically unstable upon multiple rounds of passages, which prevented amplification of the baculovirus for the large-scale AAV production. To resolve this issue, a novel rAAV vector system was developed, which contained two baculoviruses (TwoBac): one baculovirus containing the AAV gene transfer cassette and another baculovirus containing the AAV rep-cap genes together which are genetically more stable than the original system and more convenient to produce rAAV because of using TwoBac instead of three14,15. The OneBac system uses the AAV gene transfer cassette and the rep-cap genes in a single baculovirus which is more convenient to produce the rAAV because of using one baculovirus instead of using TwoBac or ThreeBac2,16,17. In our study, the TwoBac system was used for optimization.
The baculovirus system for AAV production also has limitations: baculovirus particles are unstable for long-term storage in serum-free medium11, and if the baculovirus titer is low, a large volume of baculovirus supernatant is needed, which may become toxic to the growth of Sf9 cells during AAV production (personal observation). The use of titer-less infected-cell preservation and scale-up (TIPS) cells, or baculovirus-infected insect cells (BIIC), provides a good option for AAV production in which baculovirus-infected Sf9 cells are prepared, cryopreserved, and subsequently used for infection of fresh Sf9 cells. Another advantage is the increased stability of baculovirus (BV) in Sf9 cells after cryopreservation10,11.
Two types of TIPS cells are generated to enable AAV production: the first one by infection of Sf9 cells with the BV-AAV2-GFP or therapeutic gene, and the second one by infection of Sf9 cell with BV-AAV2-rep-cap. TIPS cells are cryopreserved in small and ready-to-use aliquots. AAV vectors are produced in serum-free suspension culture in a flask placed in an orbital shaker or Wave bioreactor by co-culturing TIPS cells that produce baculoviruses and fresh Sf9 cells. Sf9 cells are infected by baculoviruses that carry the AAV2-GFP vector and the rep-cap sequences to generate AAV. Four to five days later, when AAV yields are the highest, the producer cells are lysed with detergent to release the AAV particles. The cell lysate is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the lysate by AVB Sepharose column chromatography. Finally, AAV vectors are concentrated using TFF. The protocol describes the production of AAV at a small scale, useful for research and pre-clinical studies. However, the methods are scalable and compatible with manufacturing clinical-grade AAV vectors for gene therapy applications.

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	<tbody>
		<tr >
			<td >1 N Sodium Hydroxide</td>
			<td >Sigma-Aldrich</td>
			<td >1.09137</td>
			<td >For Akta Avant cleaning</td>
		</tr>
		<tr >
			<td >2 L flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>431281</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr >
			<td >50 ml Conical tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td>For collection of supernatants</td>
		</tr>
		<tr >
			<td >24-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>07-200-80</td>
			<td>Adherent cell culture plate</td>
		</tr>
		<tr >
			<td >250 mL flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238071</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr >
			<td >Akta Avant 150 with Unicorn Software</td>
			<td>Cytiva</td>
			<td>28976337</td>
			<td>Chromatography system</td>
		</tr>
		<tr >
			<td >AVB Sepharose High Performance</td>
			<td>Cytiva</td>
			<td>28411210</td>
			<td>Chromatography medium</td>
		</tr>
		<tr >
			<td >Baculovirus-AAV-2 GFP</td>
			<td>In-house</td>
			<td>non-catalog</td>
			<td>AAV transfer vector</td>
		</tr>
		<tr >
			<td >Baculovirus-AAV-2 rep-cap</td>
			<td>In-house</td>
			<td>non-catalog</td>
			<td>AAV packaging vector</td>
		</tr>
		<tr >
			<td >Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Enzyme to degrade DNA and RNA</td>
		</tr>
		<tr >
			<td >Blocking buffer</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>516214</td>
			<td>Blocking to prevent non-specific antibody binding to cells</td>
		</tr>
		<tr >
			<td >Cell lysis buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 % Titron X-100</td>
		</tr>
		<tr >
			<td >Cellbag, 2 L and 10 L</td>
			<td>Cytiva</td>
			<td>28937662</td>
			<td>Bioreactor bag</td>
		</tr>
		<tr >
			<td >Cleaning buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>100 mM citric acid (pH 2.1)&#160;</td>
		</tr>
		<tr >
			<td >Cryovial</td>
			<td>Thomas Scientific</td>
			<td>1222C24</td>
			<td>For cryopreservation</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Growth media for cell lines</td>
		</tr>
		<tr >
			<td >Elution buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>50 mM sodium citrate buffer (pH 3.0)</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7073</td>
			<td>For disinfection and storage of the chromatography</td>
		</tr>
		<tr >
			<td >Filtration unit&#160;</td>
			<td>Pall Corporation</td>
			<td>12941</td>
			<td>Membrane filter</td>
		</tr>
		<tr >
			<td >HT1080 cell line</td>
			<td>ATCC</td>
			<td >CCL-121</td>
			<td>Fibroblast cell line</td>
		</tr>
		<tr >
			<td >HyClone&#8482; SFX-Insect culture media</td>
			<td>Cytiva</td>
			<td>SH30278.02</td>
			<td>Serum-free insect cell growth medium</td>
		</tr>
		<tr >
			<td >Peristaltic Pump</td>
			<td>Pall Corporation</td>
			<td>Non-catalog item</td>
			<td>TFF pump</td>
		</tr>
		<tr >
			<td >MaxQ 8000 orbital shaker incubator&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>Non-catalog item</td>
			<td>Shaker for suspension culture</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Nikon</td>
			<td>Non-catalog item</td>
			<td>Cell monitoring and counting&#160;</td>
		</tr>
		<tr >
			<td >Mouse anti-baculovirus gp64 PE antibody</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>65498 PE</td>
			<td>Monitoring baculovirus infection in Sf9 cells</td>
		</tr>
		<tr >
			<td >Oxygen tank</td>
			<td>Praxair</td>
			<td>Non-catalog item</td>
			<td>40 % Oxygen supply is needed for Sf9 cell growth</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>ThermoFisher Scientific</td>
			<td >20012027</td>
			<td>Wash buffer</td>
		</tr>
		<tr >
			<td >Silver Staining kit</td>
			<td >ThermoFisher Scientific</td>
			<td >LC6100</td>
			<td >Staining AAV capsid proteins</td>
		</tr>
		<tr >
			<td >Sf9 cells</td>
			<td>ThermoFisher Scientific</td>
			<td>11496-015</td>
			<td>Insect cells</td>
		</tr>
		<tr >
			<td >Steile water</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr >
			<td >Table top centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75253839/433607</td>
			<td>For clarification of Baculovirus supernatant</td>
		</tr>
		<tr >
			<td >Tangential Flow Filtration (TFF) cartridge</td>
			<td>Pall Corporation</td>
			<td>OA100C12</td>
			<td>TFF cartridge to concentrate the AAV</td>
		</tr>
		<tr >
			<td >Tris-HCl, pH 8.0</td>
			<td>ThermoFisher</td>
			<td>15568025</td>
			<td>Alkaline buffer to neutralize the eluted AAV</td>
		</tr>
		<tr >
			<td >WAVE Bioreactor System 20/50</td>
			<td>Cytiva</td>
			<td>28-9378-00</td>
			<td>Bioreactor</td>
		</tr>
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</table>

process development for the production and purification of adeno-associated virus (aav)2 vector using baculovirus-insect cell culture system

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Here, the representative results of process development for the production and purification of AAV vectors using the Sf9 insect cell system are shown. The method includes co-culture of Sf9 cells with baculovirus-infected TIPS cells, feeding the cells with growth medium, harvesting and lysis of the producer cells to release the AAV particles, clarification of the cell lysate with nuclease treatment, centrifugation and filtration, purification of AAV using AVB Sepharose affinity chromatography, and concentration with TFF (...

Watch this Scientific Journal Video about Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System at JoVE.com

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera ...

process-development-for-production-purification-adeno-associated

Research

JoVE Journal

Biology

8.9K Views.  Cincinnati Children's Hospital Medical Center. In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

Video: Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...