3.3.1. Splitting hESCs
1. Remove medium and add hESC-splitting medium to cover the well. Incubate for 1 h (most colonies will float).
2. Add 1 mL culture medium (see Subheading 2.3., items 3 or 4) and gently collect cells.
3. Collect cell suspension and place into a conical tube.
4. Centrifuge for 3 min at 90g at a recommended temperature of 4°C.
5. Resuspend cells in media and plate directly on fresh MEF-covered plates.
3.3.2. Freezing hESCs
1. Add splitting medium and incubate for 1 h.
2. Add 1 mL culture medium, gently scrape the cells, and transfer into a conical tube.
3. Do not fracture the cells into small clumps.
4. Centrifuge 3 min at 90g at a recommended temperature of 4°C.
5. Resuspend cells in culture medium (see Subheading 2.3., items 3 or 4).
6. Drop by drop, add an equivalent volume of freezing medium and mix gently (see Note 13).
7. Transfer 0.5 mL into 1-mL cryogenic vials.
8. Freeze overnight at -80°C in a Nalgene freezing box (see Note 16).
9. Transfer to liquid nitrogen on the following day (see Notes 21 and 22).
3.3.3. Thawing hESCs
1. Remove a vial from the liquid nitrogen.
2. Gently swirl the vial in a 37°C water bath.
3. When a small pellet of frozen cell remains, wash the vial in 70% ethanol.
4. Pipet the content of the vial up and down once to mix.
5. Place the content of the vial into a conical tube and add, drop by drop, 2 mL culture medium (see Subheading 2.3., items 3 or 4, and Note 17).
7. Remove the supernatant and resuspend the cells in 2 mL medium.
8. Place the cell suspension on one well of a six-well plate, or on a four-well plate, covered with MEFs.
3.3.4. Routine Culture of hESCs
1. Change the medium on a daily basis (see Note 23).
2. Passage hESCs (see Subheading 3.3.1.) every 4-6 d directly onto fresh MEF-covered plates (see Subheading 3.1.5.).
3. Scrape differentiating colonies every five to seven passages.
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