aspirate the medium. Resuspend the cells with 4 mL of ESC
medium and plate them onto feeder cells in a 60 mm dish.
Culture the cells at 37
C in a CO 2 incubator.
2. Change the medium every day and continue culturing the cells
until they become 80–90% confluent (see Note 15).
3.5 Coating of the
Culture Plate with
Poly-L-lysine and
Laminin
1. Add 75 μL of the PLL working solution to the wells of a
96-well plate (0.75 μg/well) and incubate for more than 2 h
at 37
C in a CO 2 incubator (see Note 16).
2. Add 50 μL of the laminin working solution to the wells
(0.5 μg/well) and incubate overnight at 37
C in a CO 2
incubator (see Note 17).
3.6 Differentiation
into InsulinProducing Cells
A timeline of the differentiation is shown in Fig. 1.
1. Aspirate the medium of the ESCs and wash the cells with 2 mL
of PBS. Add 1 mL of 0.25% trypsin-EDTA and incubate for
5 min at 37
C in a CO 2 incubator.
2. Dissociate the cells with a P1000 micropipette to avoid aggregation of cells and then add 4 mL of plating medium.
3. Transfer the cells to 15 mL conical tubes and count them by a
hemacytometer (see Note 18). Then, centrifuge at 200 Â g for
5 min.
4. Add a defined amount of plating medium to the cell pellet to
yield a suspension with 5.0 Â 10
4 cells/mL. Resuspend the
cells thoroughly by pipetting (see Note 19).
5. Before plating the cells, add 100 μL of plating medium to the
PLL- and laminin-coated wells; then plate 100 μL of cells into
each well to yield a density of 5000 cells/well (see Note 20).
Culture the cells for 24 h at 37
C in a CO 2 incubator.
6. One day after plating, change the medium to 200 μL of stage
1 medium (see Note 21).
7. Three days after plating, change the medium to 200 μL of stage
1 medium.
8. Five days after plating, change the medium to 200 μL of stage
1 medium. Add Pdx1 protein at a final concentration of 1 μM
(see Note 22).
9. Seven days after plating, change the medium to 200 μL of stage
2 medium. Add Pdx1 protein at a final concentration of 1 μM.
10. Nine days after plating, change the medium to 200 μL of stage
2 medium. Add NeuroD protein at a final concentration of
1 μM.
11. Eleven days after plating, change the medium to 200 μL of
stage 3 medium. Add NeuroD protein at a final concentration
of 1 μM.
92
Taku Kaitsuka and Kazuhito Tomizawa
medium and plate them onto feeder cells in a 60 mm dish.
Culture the cells at 37
C in a CO 2 incubator.
2. Change the medium every day and continue culturing the cells
until they become 80–90% confluent (see Note 15).
3.5 Coating of the
Culture Plate with
Poly-L-lysine and
Laminin
1. Add 75 μL of the PLL working solution to the wells of a
96-well plate (0.75 μg/well) and incubate for more than 2 h
at 37
C in a CO 2 incubator (see Note 16).
2. Add 50 μL of the laminin working solution to the wells
(0.5 μg/well) and incubate overnight at 37
C in a CO 2
incubator (see Note 17).
3.6 Differentiation
into InsulinProducing Cells
A timeline of the differentiation is shown in Fig. 1.
1. Aspirate the medium of the ESCs and wash the cells with 2 mL
of PBS. Add 1 mL of 0.25% trypsin-EDTA and incubate for
5 min at 37
C in a CO 2 incubator.
2. Dissociate the cells with a P1000 micropipette to avoid aggregation of cells and then add 4 mL of plating medium.
3. Transfer the cells to 15 mL conical tubes and count them by a
hemacytometer (see Note 18). Then, centrifuge at 200 Â g for
5 min.
4. Add a defined amount of plating medium to the cell pellet to
yield a suspension with 5.0 Â 10
4 cells/mL. Resuspend the
cells thoroughly by pipetting (see Note 19).
5. Before plating the cells, add 100 μL of plating medium to the
PLL- and laminin-coated wells; then plate 100 μL of cells into
each well to yield a density of 5000 cells/well (see Note 20).
Culture the cells for 24 h at 37
C in a CO 2 incubator.
6. One day after plating, change the medium to 200 μL of stage
1 medium (see Note 21).
7. Three days after plating, change the medium to 200 μL of stage
1 medium.
8. Five days after plating, change the medium to 200 μL of stage
1 medium. Add Pdx1 protein at a final concentration of 1 μM
(see Note 22).
9. Seven days after plating, change the medium to 200 μL of stage
2 medium. Add Pdx1 protein at a final concentration of 1 μM.
10. Nine days after plating, change the medium to 200 μL of stage
2 medium. Add NeuroD protein at a final concentration of
1 μM.
11. Eleven days after plating, change the medium to 200 μL of
stage 3 medium. Add NeuroD protein at a final concentration
of 1 μM.
92
Taku Kaitsuka and Kazuhito Tomizawa