References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… hESC Cell Culture

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Lab Week 1 Overview Tuesday (1.5h) – Lab safety and finding your way around the lab – Learn how to use pipettes, incubators, biosafety cabinets, microscopes – Prepare media and tissue culture dishes and thaw feeder cells Thursday (1.5h) – Pre-warm media – Check feeder cell plates thawed on Tuesday – Thaw and plate stem cells 2

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Thawing hESCs: 1 Be sure to have a 6 well with “hESC-MEFs” available. 2 Add 3+9 ml hESC medium to two 15 ml tubes and pre-warm to 37C. 3 Hold frozen hESC vial in 37C water bath (do not immerse) until a small piece of ice remains, then quickly ethanol-spray and wipe off vial and move to hood. 4 Transfer a small amount of warm medium from the 9 ml falcon tube to the hESC tube using a 1 ml serological plastic pipette and resuspend hES cell clumps by slowly pipetting up and down (once only!). 5 Transfer to the 9 ml falcon tube. 6 Spin. 7 Aspirate supernatant above the MEFs, then wash this well with 2 ml of DMEM/F12 and 8 add 1 ml of hESC medium from the 3 ml tube. 9 Remove the medium above the spun hESCs. 10 Carefully resuspend pelleted cell clumps using a 2 ml serological plastic pipette and the remaining 2 ml of fresh hESC medium (pipette just enough to resuspend and evenly mix the cell clumps in the medium) 9 Plate onto the MEF well. 10 Transfer to the incubator (rock the plate back/forth and left/right, avoiding circular motions, to ensure even plating)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… hESCs: Healthy Culture MEFs Colony ~40xPh ~4xPh hESC (nucleus) nucleoli (dark-field) Look for: tight borders Look for: tightly packed stem cells (phase-contrast) 4

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… dying/dead cells and cellular debris (some of this stuff will be floating in the media as well!) hESC inter-cellular spaces … may be essentially invisible (typical for center of large colonies) … may appear “phase-bright” (typical for smaller colonies and colony periphery) these are still good stem cells! (despite the difference in size, shape, and appearance) 5 hESCs: Healthy Culture

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… ~1 day after plating ~3 days after plating time to split again! 6 hESCs: Colony Growth