# AAV production with HEK293T

```{note}
This page is still under active development
```

## Cell lines

> AAV 293 or HEK293T

## Materials & Reagents

- Cell
- DMEM (high glucose without L-glutamine)
- Fetal bovine serum (FBS)
- TrypLE Express
- Penicillin/Streptomycin (P/S)
- AdDeltaF6 (Addgene, [#112867](https://www.addgene.org/112867/))
- AAV2/1 (Addgene, [#112862](https://www.addgene.org/112862/))
- AAV2/1 (Addgene, [#104964](https://www.addgene.org/104964/))
- AAV retrohelper (Addgene, [#81070](https://www.addgene.org/81070/))
- Transfer plasmid (ITR-ITR)
- Lipofectamine 3000
- Opti-MEM
- AAVpro Purification Kit Midi (All Serotypes) Takara
- 0.5 M EDTA (pH 8.0)

Culture medium
: DMEM +10% FBS + 1%P/S

| Reagent | Volume |
|:-------:|:------:|
|  DMEM   | 445 ml |
|   FBS   | 50 ml  |
|   P/S   |  5 ml  |
|         |        |
|  Total  | 500 ml |

---

## Protocols

### Thawing cells from freezing cells``

1. Thaw AAV293 cells in 37°C waterbath.
2. Add to 5 mL of 293T culture medium.
3. Spin down for 5 min at 1000 rpm
4. Resuspend the cells with 10 mL 293T culture medium.
5. Plate in 150 cm culture dish.

```{important}
Change medium every day or every two days. If you don’t and the medium gets yellow, the cells will detach and die.
```

6. Once the cells are 90% confluent, split them 1 to 4 using TrypLE express:
7. Rince with DPBS--
8. Add 5 ml of TrypLE express and incubator for 5-7 min
9. Spin down for 5 min at 1000 rpm, then aspirate supernatant
10. Resuspend in 8 mL medium per flask.
11. Plate 2 mL cell suspension + 8 mL of fresh medium per flask to split ¼.

### Transfection

### Collection Option 1

```{important}
The protocol below describes purification of AAV particles from producer cells in one T225 flask or four 10 cm dishes.
```

```{seealso}
https://takara.co.kr/file/manual/pdf/6675_6675S_e.v2001Da.pdf
```

#### VI-1 Preparation of AAV extract solution

1. Detach the AAV-producing cells by adding 1/80 volume of 0.5 M EDTA (pH 8.0)
to the culture medium; incubate at room temperature for 10 min.
2. Collect the cells in a centrifuge tube from the flask.
3. Centrifuge at 1,700 - 2,000g for 10 min at 4℃ and discard the supernatant.
4. Centrifuge again at 1,700 - 2,000g for 1 min at 4℃ and remove the supernatant completely.

```{note}
Be sure to completely remove the supernatant, because remaining
supernatant could impair virus purification.
```

5. Loosen the cell pellet well by tapping or vortexing.

```{note}
If the cell pellet is not loosened sufficiently, the purification efficiency can be decreased. Make sure there are no cell clumps before proceeding to the next step.
```
6. Add 2 ml of AAV Extraction Solution A plus.
7. Resuspend by vortexing for 15 sec.

```{note}
Continue to vortex until there are no remaining cell clumps.
```

8. Incubate for 5 min at room temperature and then vortex again for 15 sec
9. Centrifuge at 4,000 - 9,000g for 10 min at 4℃
```{note}
In some cases, the virus recovery can be improved by repeating steps 7 - 9.
```
10. Transfer the supernatant in a new sterile centrifuge tube using a pipet to avoid contamination. Add 1/10 volume of AAV Extraction Solution B to the
supernatant.

```{note}
The virus suspension can be stored at -80℃. Alternatively, promptly proceed to step VI-2-1. 
If the virus suspension is stored at -80℃, thaw in a 37℃ incubator before using. Be sure to use a tube that is resistant to freezing and centrifugation when a virus suspension is stored at -80℃.
```

```{note}
When AAV Extraction Solution B is added, the color of the solution may turn pink in some cases; this does not affect performance.
```

#### VI-2. Purification and concentration of AAV particles

``Purification``

```{note}
Use swing bucket rotors for steps VI-2-5, VI-2-6, and VI-2-7.
```

1. Add 1/100 volume of Cryonase Cold-active Nuclease to the virus suspension at step VI-1-10, and then incubate at 37℃ for 1 hr.
2. Add 1/10 volume of Precipitator A, vortex for 10 sec, incubate at 37℃ for 30 min, and vortex again for 10 sec.

```{note}
Precipitator A may produce a white precipitate at low temperature, however this does not affect the quality or performance of this reagent. If a precipitate is present, dissolve it completely at 37℃
before use.
```

```{note}
Although a precipitate may form during the incubation, this is not
a problem. Proceed to the next step.
```

3. Add 1/20 volume of Precipitator B to the mixture at the step above, vortex quickly for 10 sec, and then centrifuge at 5,000 - 9,000g for 5 min at 4℃.

```{note}
A precipitate may be formed after adding Precipitator B, but proceed to centrifugation.
```

4. Filter the supernatant using Millex-HV 0.45 μm.
5. Transfer the filtrate containing AAV vector into a filter device of Amicon Ultra-4, 100 kDa. Centrifuge at 2,000g for 5 min at 15℃, and then confirm that the AAV solution in the filter device is <0.4 ml.

```{note}
If the volume of the solution is >0.4 ml, continue to centrifuge.
```

6. After removing the filtrate, add 1 ml of Suspension Buffer in the filter device of the Amicon Ultra-4 and mix the solution uniformly by pipetting. Centrifuge at 2,000g for 5 min at 15℃, and then confirm that the AAV solution inside the filter device device is <0.4 ml.

```{note}
If the volume of the solution is >0.4 ml, continue to centrifuge.
```

7. Repeat step VI-2-6 4 times (total 5 times) to obtain an appropriate volume of solution.
8. After discarding the filtrate, resuspend the solution inside of the cup of the Amicon Ultra-4, 100 kDa filter device by pipetting or vortexing for 30 sec and transfer the virus suspension to a new tube.

### Option 2

####