# iPS cell culture

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

## Cell lines
>
> Tatsuya's cell line list

## Reagent

- iPS cells
- mTeSR plus (Stem cell technolgies, culture meidum)
- Matrigel ESC qualified
- ReLeSR™ Human PSC Selection & Passaging Reagent (Stem cell technolgies,)
- Y-27632 (Rock inhibitor)
- CryoStor CS10 | freezing solution  (Stem cell technolgies,)

## Protocols

```{seealso}
[Document of mTeSR Plus for cell culture protocol](https://cdn.stemcell.com/media/files/manual/MADX23032-Maintenance_of_Human_Pluripotent_Stem_Cells_in_mTeSR_Plus.pdf)
```

``Thawing cells from freezing cells``

1. Thawing matrigel stock solution on ice (1.5 ml tube)
2. Add __cold__ 10ml of DMEM/F12 into 15ml of falcon tube.

```{important}
Must use cold DMEM/F12
```

1. Add Matrigel solution into prepared 10ml of DMEM/F12 in 15 ml falcon tube
2. Coat 6 well late with Matrigel , and incubator for one hour at RT
   - 6 well plate: 1 mL per well

```{important}
Do not put into incucator
```

3. Thawing the cells in freezing vial (from LN2 Tank) at 37C water bath
4. Add 4 ml of mTeSR plus new 15 ml falcon tube.
5. Add 1 ml of cell suspention from freezing vial into 15 ml of falcon tube.
6. Centrifuge 15ml of falcon tube at 1000 rpm for 3 min at RT.
7. Aspirate supernatant
8. Add 2 ml of fresh mTeSR plus into 15 ml of falcon tube containing cell pelet. Pipette and mix for several times.

```{important}
Do not mix more than 5 times, use 5 ml of pipette
```

9. Transfer cell suspension to 6 well plate
10. Place 6 well plate into incubator.

``Medium change``

1. Aspirate mTeSR plus from 6 well plate
2. Add 2 ml of mTeSR plus into 6 well plate

``Subculture``

1. Aspirate culture medium from 6 well plate
2. Add 1ml of ReLeSR™ into 6 well plate
3. Aspirate ReLeSR™ from 6 well plate
4. Incubate the plate for 3-5 min
5. Add flesh 1 ml of mTeSR plus, and then tapping the plate for 10-30 sec until you can see floating cells and to get 30-50 um of cell colony.
6. collect all soulstion into a 15mL falcon tube.
7. Add 7 ml of mTeSR plus into falcon tube.
8. 2 ml of cell suspension into Matrigel coatd 6 well plate
9. Place 6 well plate on incubator.

---