iPSC culture (mTeSR1)

Protocol iPSC culture for chimpanzee iPSCs and ZNF492-WT and KO cells

Coating of plates

• thaw Geltrex on ice
• dilute Geltrex in cold DMEM/F12 1:100 (for 1 well of a 6-well plate 1 ml diluted Geltrex is needed)
• incubate for 1 h. at RT

Thawing of iPS cells

• coat plates with Geltrex
• prewarm mTeSR1 medium
• prepare 4 ml mTeSR1 in 15 ml falcon tube
• thaw cells at 37°C
• move cells to prepared falcon tube
• centrifuge for 5 min
• remove Geltrex from coated plates and add 1 ml mTeSR1 per well
• remove supernatant
• resuspend cells in 1 ml mTeSR1 using a 5 ml serological pipette
• move cells to coated plate
• incubate at 37°C and 5% CO2
• daily medium changes

Medium Change

• prewarm mTeSR
• remove old medium
• add 2 ml mTeSR1
• incubate at 37°C and 5% CO2

Passaging of iPS cells

• coat plates with Geltrex
• prewarm mTeSR1 medium
• wash cells with PBS
• add 1 ml ReLeSR (at RT)
• remove ReLeSR
• incubate in incubator for 3 min.
• add 1 ml mTeSR1 and tap for 30 sec.
• move cells to a falcon tube and add 5 ml mTeSR1 for 1:6 split
• remove Geltrex from the coated plates and add 1 ml mTeSR1 per well
• move 1 ml cell suspension to each well of the plate
• incubate at 37°C and 5% CO2
• daily medium changes

Freezing of iPS cells

• thaw mFreSR1
• wash cells with PBS
• add 1 ml ReLeSR (at RT)
• remove ReLeSR
• incubate in incubator for 3 min.
• add 1 ml mFreSR1 and tap for 30 sec.
• move cell suspension to a cryotube
• freeze in a Mr. Frosty at -80°C