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

See also

Document of mTeSR Plus for cell culture protocol

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.

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