Table 5.

Colon differentiation medium composition

Component	Stock concentration	Volume added	Final concentration	Recommended storage condition
R-spondin 1 conditioned media	100 ×	2 ml	20 ×	−20 °C freezer
Base media*	100 ×	8 ml*	80 ×	4 °C fridge
B27	50 ×	200 μl	1 ×	−20 °C freezer
N2	100 ×	100 μl	1 ×	−20 °C freezer
N-acetyl cysteine	500 mM	10 μl	500 μM	−20 °C freezer
A83-01	500 μM	10 μl	500 nM	−20 °C freezer
Noggin	100 μg/ml	10 μl	100 ng/ml	−80 °C freezer
EGF	250 μg/ml	2 μl	50 ng/ml	−20 °C freezer
human [Leu15]-Gastrin	100 μM	1 μl	10 nM	−20 °C freezer
Final volume		10.03 ml

^*Base media consists of Advanced DMEM/F12 (Gibco, 12634-01), 2mM Glutamax, 10 mM HEPES, 100 units/ml. Penicillin/Streptomycin. For antibiotic-free culture, penicillin/streptomycin is excluded, as described in Table 1.