Editing AAV-Production-293T-2 (section)

== AAV stock production <br>  ==

1. Thaw a vial of 293T cells (each vial contains ~3 Ã 10^6 cells) and plate cells in a 10-cm dish containing 10 ml D10 (DMEM with 10% FBS). Maintain in a 37 °C incubator with 5% CO2 atmosphere.<br> 

<br>2. After 3 days, aspirate the medium and wash the dish with 2 ml of 1Ã PBS. Add 2 ml of trypsin-EDTA solution and incubate at 37 °C for 2 min or until cells detach from culture dish.<br> 

<br>3. Add 4 ml of D10 to neutralize trypsin-EDTA solution and transfer the cell suspension to a 15-ml conical tube.<br> 

<br>4. Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.<br> 

<br>5. Resuspend the cells in 1 ml of D10. Take a 10-ÎŒl aliquot and count the cells using a hemocytometer.<br> 

<br>6. Adjust cell concentration to 10^6 cells per ml with D10 and plate 1 ml of cell suspension in eight 10-cm dishes, each contains 10 ml D10. Culture for 72 h in a 37 °C incubator with 5% CO2 atmosphere.<br> 

<br>7. After the 3-d incubation, harvest cells as described in Steps 2â4. Resuspend in 5 ml of D10 and take a 10-ÎŒl aliquot for counting with a hemocytometer. Adjust cell concentration to 4 Ã 10^6 cells per ml and plate 1 ml of cell suspension in 30 10-cm dishes, each containing 10 ml D10. Incubate in a 37 °C incubator overnight.<br> 

<br>8. The next day, prepare solution A by combining 15.5 ml of 2Ã HEPES-saline and 155 ÎŒl of 0.15 M phosphate buffer in a 50-ml tube and, mixing well.<br> 

<br>9. In another 50-ml tube, prepare solution B by combining 1.938 ml of 2 M CaCl2, 387.5 ÎŒg of plasmid.<br> 

<br>10. Add solution B to solution A, mix well and quickly add 1.0 ml of the transfection mix to each of the 30 dishes of 293T cells.<br> Use a 10-ml pipette and tilt the dish so that the 1.0 ml of transfection mix is added to a greater depth of medium to prevent âblastingâ cells off the dish.<br> 

<br>11. Agitate the dishes gently to evenly distribute the precipitate and return the dishes to the 37 °C incubator.<br> 

<br>12. Observe a dish 1 h later under a microscope. The precipitate should be visible as small grains about the size of bacteria. Return the dishes to the 37 °C incubator.<br> 

<br>13. After 48 h, prepare dry ice/ethanol bath by placing a centrifuge tube rack in a styrofoam container. Add ~2 lbs of dry ice, pour 2 liters of ethanol over it and cover the container.<br> 

<br>14. Collect the cells and medium by scraping the cells off the dish with a rubber policeman and transferring it to a 50-ml conical tube. Use Corning 50-ml conical tubes, as they are more resistant to the subsequent freeze-thaw cycles. Rinse dishes with 5 ml of 1Ã PBS and transfer it to the same conical tube. Harvest four dishes at a time into the same 50-ml tube.<br> 

<br>15. Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.<br> 

<br>16. Aspirate the medium from the conical tube and repeat Steps 14 ~ 15 until cells are pelleted from all 30 dishes. The same tube can be used for pelleting cells from all the dishes.<br> 

<br>17. Resuspend the final pellet from the 30 10-cm dishes in 8 ml of cell lysis buffer.<br> 

<br>18. Freeze the pellet in the dry ice/ethanol bath and thaw in a 37 °C water bath.<br> 

<br>19. Repeat Step 18 twice and store at â 80 °C.<br> 

<br> The pellet can be stored at â 80 °C indefinitely and thawed at your convenience.<br> 

<br>20. Thaw cell lysate in a 37 °C water bath.<br> 

<br>21. Add Benzonase to the cell lysate at a final concentration of 50 U ml â 1. Benzonase is added to eliminate cellular DNAs/RNAs as well as excess plasmid DNAs present during transfection.<br> 

<br>22. Incubate at 37 °C in a water bath for 30 min. Prepare iodixanol gradient during the incubation.<br> 

<br>23. Centrifuge at 5,000g for 30 min at 4 °C in a Sorvall high-speed centrifuge using an HS-4 rotor.<br> 

<br>24. Collect vector-containing supernatant. The volume of the supernatant is approximately 5 ml.<br> 

<br>25. Load the 5 ml of vector-containing supernatant over the iodixanol density gradient prepared in Step 22. Top off the tube with cell lysis buffer. This density gradient purification step can remove contaminants that may influence transduction and targeting frequencies.<br> 

<br>26. Centrifuge at 461,300g for 1 h at 18 °C with maximum acceleration and deceleration in a Beckman Ti70 rotor on a Beckman ultracentrifuge; use proper spacers for tubes.<br> 

<br>27. Puncture the tube on the side slightly below (3â5 mm) the 60â40% interface with an 18-gauge needle (bevel up) attached to a 10 ml syringe.<br>28. Collect 3â4 ml from each centrifuge tube by aspiration using the same needle.<br> 

<br>CRITICAL STEP Avoid the proteinaceous material near the 40â25% interface.<br> 

<br>29. To use the vector without removing iodixanol, proceed directly with Step 30; iodixanol is inert and non-toxic to cells. Alternatively, further purify Serotype 2 vector stocks over a HiTrap heparin affinity column, followed by desalting over a HiTrap desalting column as described in Box 4, before proceeding with Step 30.<br> 

<br>