Protocols

Monitoring Morphology of Colonies

The hES cell cultures require daily monitoring of morphology and a daily change of media.

The first robust colonies of hES are observed 48 hrs after plating – they are provided to you 24 hrs after plating. hES cell colonies appear as a single layer and translucent discs of about 100 to 300 cells under low power magnification . Exponential growth occurs for 2-3 days following plating. The edges of  these hES cell colonies are smooth and  show a morphologic feature of undifferentiated cells. Growth rates decline at day 5-6 when colonies reach 400 cells.

Colonies larger than 500 cells increase the risk of differentiation and karyotypic instability. Therefore we recommend that your experiments are planned carefully for treatments at the appropriate times.

When large colonies are detected, this requires mechanical disruption and replating on a fresh MEF feeder layer to prevent differentiation. Early signs of differentiation include protrusions from one colony to the adjacent colony resulting in contact between neighboring colonies, as well as loss of translucency. Sections or whole colonies appear darker by light microscopy due to the build-up of multiple cell layers.