HESC Culture

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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