2 mM L-Gln, 1 mM sodium pyruvate, 50 U/mL penicillin,
50 μg/mL streptomycin, and 100 μM β-mercaptoethanol
(β-ME). Store at 4
C.
2.3 Differentiation
into InsulinProducing Cells
Make all media, buffers, and solutions sterile.
1. Plate: 96-well plate with an Ultra-Web synthetic surface (Corning) or polyamide net-like fibers (Japan Vilene Company, Ltd.)
(see Note 2).
2. Phosphate-buffered saline (PBS).
3. 1 mg/mL PLL stock solution: Dissolve 1 mg of poly-L-lysine
in 1 mL of sterile PBS. Store at 4
C.
4. 10 μg/mL PLL working solution: Mix 0.1 mL of PLL stock
solution with 9.9 mL of sterile PBS. Store at 4
C.
5. 10 μg/mL Laminin working solution: Mix 0.1 mL of laminin
solution (laminin-111 from mouse Engelbreth-Holm-Swarm
sarcoma; Roche) with 4.9 mL of sterile DMEM.
6. Plating medium: high-glucose (4500 mg/L) DMEM supplemented with 10% FBS, 100 μM NEAAs, 2 mM L-Gln, 50 U/
mL penicillin, 50 μg/mL streptomycin, and 100 μM
β-ME. Store at 4
C.
7. 0.2 g/mL AlbuMAX II stock solution: Add approximately
100 mL of water to a glass beaker. Weigh 25 g of AlbuMAX
II (Thermo Fisher Scientific) and transfer to the beaker. Mix
and make up to 125 mL with water. Sterilize through a 0.22-μ
m filter. Store at À20
C.
8. 100 μg/mL Activin A stock solution: Add 100 μL of sterile
0.1% bovine serum albumin (BSA)-PBS to a vial containing
10 μg of Activin A (R&D Systems) and mix. Store at À80
C.
9. 50 μg/mL Basic fibroblast growth factor (bFGF) stock solution: Add 1 mL of sterile 0.1% BSA-PBS to a vial containing
50 μg of bFGF (PeproTech) and mix. Store at À80
C.
10. 100 mM Retinoic acid (RA) stock solution: 100 mM RA in
sterile dimethyl sulfoxide (DMSO). Store at À20
C.
11. 250 μM Cyclopamine stock solution: 250 μM KAADcyclopamine (EMD Millipore) in sterile DMSO. Store at
À20
C.
12. 50 μg/mL Fibroblast growth factor 10 (FGF10) stock solution: Add 0.5 mL of sterile 0.1% BSA-PBS to a vial containing
25 μg of FGF10 (PeproTech) and mix. Store at À80
C.
13. 1 M Nicotinamide (NAM) stock solution: 1 M in PBS. Sterilize
through a 0.22-μm filter. Store at À20
C.
14. 100 μM Glucagon-like peptide-1 (GLP-1) stock solution:
100 μM in sterile PBS. Store at À80
C.
88
Taku Kaitsuka and Kazuhito Tomizawa
50 μg/mL streptomycin, and 100 μM β-mercaptoethanol
(β-ME). Store at 4
C.
2.3 Differentiation
into InsulinProducing Cells
Make all media, buffers, and solutions sterile.
1. Plate: 96-well plate with an Ultra-Web synthetic surface (Corning) or polyamide net-like fibers (Japan Vilene Company, Ltd.)
(see Note 2).
2. Phosphate-buffered saline (PBS).
3. 1 mg/mL PLL stock solution: Dissolve 1 mg of poly-L-lysine
in 1 mL of sterile PBS. Store at 4
C.
4. 10 μg/mL PLL working solution: Mix 0.1 mL of PLL stock
solution with 9.9 mL of sterile PBS. Store at 4
C.
5. 10 μg/mL Laminin working solution: Mix 0.1 mL of laminin
solution (laminin-111 from mouse Engelbreth-Holm-Swarm
sarcoma; Roche) with 4.9 mL of sterile DMEM.
6. Plating medium: high-glucose (4500 mg/L) DMEM supplemented with 10% FBS, 100 μM NEAAs, 2 mM L-Gln, 50 U/
mL penicillin, 50 μg/mL streptomycin, and 100 μM
β-ME. Store at 4
C.
7. 0.2 g/mL AlbuMAX II stock solution: Add approximately
100 mL of water to a glass beaker. Weigh 25 g of AlbuMAX
II (Thermo Fisher Scientific) and transfer to the beaker. Mix
and make up to 125 mL with water. Sterilize through a 0.22-μ
m filter. Store at À20
C.
8. 100 μg/mL Activin A stock solution: Add 100 μL of sterile
0.1% bovine serum albumin (BSA)-PBS to a vial containing
10 μg of Activin A (R&D Systems) and mix. Store at À80
C.
9. 50 μg/mL Basic fibroblast growth factor (bFGF) stock solution: Add 1 mL of sterile 0.1% BSA-PBS to a vial containing
50 μg of bFGF (PeproTech) and mix. Store at À80
C.
10. 100 mM Retinoic acid (RA) stock solution: 100 mM RA in
sterile dimethyl sulfoxide (DMSO). Store at À20
C.
11. 250 μM Cyclopamine stock solution: 250 μM KAADcyclopamine (EMD Millipore) in sterile DMSO. Store at
À20
C.
12. 50 μg/mL Fibroblast growth factor 10 (FGF10) stock solution: Add 0.5 mL of sterile 0.1% BSA-PBS to a vial containing
25 μg of FGF10 (PeproTech) and mix. Store at À80
C.
13. 1 M Nicotinamide (NAM) stock solution: 1 M in PBS. Sterilize
through a 0.22-μm filter. Store at À20
C.
14. 100 μM Glucagon-like peptide-1 (GLP-1) stock solution:
100 μM in sterile PBS. Store at À80
C.
88
Taku Kaitsuka and Kazuhito Tomizawa