Example 1
Proliferation of Pluripotent and/or Multipotent Stem Cells
When the cells have reached about 90% confluency the cells were splitted. For that the medium was removed and the cells were washed with PBS and then harvested with TrypLE (Life Technologies, 12563029). The cell suspension was centrifuged for 5 min at 300×g. The supernatant was removed and diluted in fresh StemMACS iPS-Brew XF medium (Miltenyi Biotec, 130-104-368). After determining the cell number 80.000-100.000 cells per 6-well were seeded in 2 ml fresh iPS Brew medium with Thiazovivin (2 μM; Miltenyi Biotec 130-104-461) in 6-well plates which were coated with Laminin-521 (0.5 μg/cm2; BioLamina LN521-02) diluted in PBS++ over night at 4° C. After two days the medium was changed on daily basis without Thiazovivin until reaching about 90% confluency.
