Animal Free Culturing

3.2.1. Derivation of Foreskin Fibroblast Lines (see Note 13)

1. Place a human foreskin in PBS supplemented with penicillin-streptomycin (see Note 14).

2. Unfold the foreskin and wash three times with PBS.

3. Cut into small pieces using sharp Iris scissors (approximately eight pieces per foreskin).

4. Transfer the clean pieces into a new Petri dish and mince thoroughly using sharp Iris scissors.

5. Add 6 mL trypsin/ethylenediaminetetraacetic acid (EDTA) and incubate for at least 30 min.

6. Neutralize the trypsin using at least 6 mL of foreskin fibroblasts culture medium (see Subheading 2.2., item 2). Transfer the foreskin fibroblasts into conical tubes and use foreskin fibroblast culture medium to wash the plate.

7. Divide evenly into T-25 culture flasks at a recommended ratio of three pieces per flask.

8. Add 6 mL foreskin fibroblast culture medium.

9. Grow the foreskin fibroblasts until confluent culture is achieved. Change the medium as needed (do not vacuum the lumps).

10. Freeze the resultant foreskin fibroblasts to create a vial bank of the line (see Subheading 3.4.).

3.2.2. Splitting Foreskin Fibroblasts

1. Split foreskin fibroblasts every 5-7 d (see Note 15) by adding 2 mL trypsin/EDTA to cover the entire culture-flask surface.

2. Incubate for 6 min.

3. Tap the side of the flask to loosen the cells and add 4 mL of culture medium (see Subheading 2.2., item 2) to neutralize the trypsin.

4. Remove the cell suspension into a conical tube and centrifuge for 5 min at 300g.

5. Remove the suspension, resuspend in culture medium, and pipet to fracture the pellet.

6. Distribute the cell suspension to a desired number of culture flasks (recommended splitting ratio is 1:3).

7. Add 6 mL of foreskins fibroblast culture medium (see Subheading 2.2., item 2).

3.2.3. Freezing Foreskin Fibroblasts

1. Remove all lumps as much as possible.

2. Add 2 mL trypsin EDTA and cover the entire culture-flask surface.

3. Incubate for 6 min.

4. Tap the side of the flask to loosen the cells and add 4 mL of culture medium (see Subheading 2.2., item 2) to neutralize the trypsin.

5. Remove the cell suspension into a conical tube. Let the remaining lumps sink and remove the cell suspension into a clean conical tube.

6. Centrifuge for 5 min at 300g.

7. Add culture medium and pipet up and down to break the cell pellet.

8. Drop by drop, add an equivalent volume of freezing medium (see Subheading 2.1., items 1 or 2) and mix gently (see Note 10).

9. Place 1 mL into 2-mL cryogenic vials (a recommended freezing ratio of one or two vials per confluent flask).

10. Freeze the vials overnight at -80°C in a Nalgene freezing box (see Note 11).

11. Transfer the vials into a liquid nitrogen container (see Note 12).

3.2.4. Thawing Foreskin Fibroblasts

1. Remove the vial from the liquid nitrogen and quickly thaw it in a 37°C water bath.

2. When a small pellet of frozen cells remains, clean the vial using 70% ethanol.

3. Pipet the content of the vial up and down once and transfer the cells into a conical tube.

4. Drop by drop, add 2 mL of culture medium (see Subheading 2.2., item 2, and Note 9).

5. Centrifuge for 5 min at 300g.

6. Resuspend the pellet in culture medium.

7. Remove the cell suspension into culture flasks and add 6 mL culture medium.

3.2.5. Preparation of Foreskin Fibroblast-Covered Plates

1. Add 8 |g/mL mitomycin C into a culture flask and incubate for 2 h.

2. Wash four times with PBS.

3. Add 2 mL of trypsin EDTA and cover the entire culture-flask surface.

4. Incubate for 6 min.

5. Tap the side of the flask to loosen the cells and add 4 mL of culture medium (see Subheading 2.2., items 3 or 4) to neutralize the trypsin.

6. Remove the cell suspension into a conical tube.

7. Centrifuge for 5 min at 300g.

8. Add 10 mL of culture medium (see Subheading 2.2., items 3 or 4) and pipet up and down to break the cell pellet.

9. Count the cells and resuspend them in the desired medium volume.

10. Add the cell suspension into the culture dishes at a plating density of 4 x 105 cells per well in six-well plates (10 cm2).

11. Let set for at least 3 h before plating the hESCs.

3.2.6. Routine Culture of hESCs With Foreskin Fibroblasts

1. Passage hESCs (see Subheading 3.1.1.) every 4-6 d directly on foreskin fibroblast-covered plates (see Subheading 3.2.5.).

2. Change the medium (see Subheading 2.2., items 3 or 4) on a daily basis.

3. Scrape differentiating colonies every five to seven passages.

4. Freeze the cells and thaw as described in Subheadings 3.1.2. and 3.1.3., respectively.

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