3.1 Human iPSC Culture

For the coating of cell culture plates, resuspend one aliquot of Matrigel in the appropriate amount of cold coating medium ( see Note 1 ). Add the Matrigel solution to the cell culture plates and distribute equally so that the entire well is covered ( see Note 6 ).

Incubate at Room temperature for at least 0h 45m 0s. Use immediately or store at 4°C for a maximum of 2 weeks. Prior to use, simply aspirate the coating medium and add the cell suspension. No washing step is required.

To thaw iPSCs, get the frozen vials from the liquid nitrogen tank and keep on dry ice. Thaw carefully in a 37°C water bath or alternatively with the ThawSTAR™ Automated Cell Thawing System (BioCision™) until only a small ice cube remains.

Transfer the cell solution to a 15 ml Falcon tube and add dropwise 2mL–3mL.

Spin down at 400x g and aspirate the supernatant.

Resuspend the cell pellet in mTeSR™1 with ROCKi and transfer to a cell culture plate coated with Matrigel and place in the incubator (37°C, 5%).

After 24h 0m 0s, wash the cells once with 1x and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until the next passaging ( see Note 7 ).

In order to passage iPSCs, aspirate the culture medium and wash the cells once with 1x. Dissociate the cells by adding TrypLE and place in the incubator for approximately 0h 2m 0s–0h 3m 0s.

Add 1x and pipet up and down to collect all cells.

Transfer the cell solution to a 15 ml Falcon tube and spin down at 400x g. Aspirate the supernatant and resuspend the cell pellet in mTeSR™1 with ROCKi.

Count the cells using Trypan Blue (e.g., with the Countess™ II FL Automated Cell Counter or hemocytometer) and seed the appropriate number of cells in Matrigel- or poly-L-lysine + laminin-coated cell culture plates ( see Note 8 ). Mix well and place in the incubator (37°C, 5%).

After 24h 0m 0s, wash the cells once with 1x and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until next passaging ( see Note 7 and Fig. 1). It is recommended to check the iPSCs regularly for mycoplasma contamination ( see Note 9 ).

To freeze iPSCs, dissociate the cells with TrypLE and collect in 1x, spin down and resuspend the pellet in mFreSR™ medium. If necessary, count the cells and aliquot the appropriate amount into cryotubes ( see Note 10 ).

Put the tubes in a freezing container and store at -80°C for at least 2h 0m 0s. Subsequently, store in liquid nitrogen.