3.1.1. Splitting hESCs
The following protocol is suitable for hESCs cultured either with foreskin fibroblasts or in feeder-free conditions using fibronectin-covered plates.
1. Remove medium from well. Add 0.5 mL (for six-well plates) splitting medium (see Subheading 2.1., item 3) and incubate for 1 h (most colonies will float).
2. Add 1 mL of culture medium and gently collect cells with a 5-mL pipet (differentiated cells will remain on the plate).
3. Collect cell suspension and place into a conical tube.
4. Centrifuge 3 min at 90g at a recommended temperature of 4°C.
5. Resuspend cells in media and plate directly on a ready-to-use culture plate.
The following protocol can be used for hESCs cultured either with foreskin fibroblasts or in feeder-free conditions using fibronectin-covered plates. The recommended freezing ratio is 10 cm2 per vial (one well in six-well plates).
1. Add splitting medium to plate and incubate for 1 h.
2. Add 1 mL culture medium, gently scrape the cells using a 5-mL pipet and transfer into a conical tube (see Note 9).
4. Resuspend cells in a culture medium (see Subheading 2.2., items 3 or 4).
5. Drop by drop, add an equivalent volume of freezing medium (either Subheading 2.1., items 1 or 2) and mix gently (see Note 10).
6. Pour 0.5 mL into 1-mL cryogenic vials.
7. Freeze overnight at -80°C in a freezing box (see Note 11).
8. Transfer to liquid nitrogen on the following day (see Note 12).
3.1.3. Thawing hESCs
This method can be used for hESCs culture with either foreskin fibroblasts or in a feeder-free environment.
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 of culture medium (see Note 10).
7. Remove the supernatant and resuspend the cells in 2 mL of medium.
8. Place the cell suspension on one well of a six-well plate (or on a four-well plate) precovered with fibronectin (see Subheading 2.3., item 1) or foreskin fibroblasts.
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