Table 3.

Colon organoid growth medium composition

Component	Stock concentration	Volume added	Final concentration	Recommended storage condition
WRN-conditioned media	100 ×	6.5 ml	65 ×	−20 °C freezer
Base media	100 ×	3.2 ml*	32 ×	4 °C fridge
B27	50 ×	200 μl	1 ×	−20 °C freezer
Nicotinamide	1 M	100 μl	10 mM	−20 °C freezer
N2	100 ×	100 μl	1 ×	−20 °C freezer
N-acetyl cysteine	500 mM	10 μl	500 μM	−20 °C freezer
A 83-01	500 μM	10 μl	500 nM	−20 °C freezer
Y-27632 dihydrochloride	10 mM	10 μl	10 μM	−20 °C freezer
SB 202190	30 mM	3.33 μl	10 μM	−20 °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
Prostaglandin E2	10 mM	2 μl**	5 nM	−80 °C freezer
Final volume		10.14 ml

^*Base media consists of Advanced DMEM/F12, 2 mM Glutamax, 10 mM HEPES, 100 units/ml penicillin/streptomycin, described in Table 2. WRN: Wnt, R-spondin 1, Noggin. EGF: epidermal growth factor.

^**Prostaglandin E₂ should be diluted before adding. Make a dilution of 1 μl PGE stock to 349 μl BM. Add 2 μl PGE dilution for 10 ml media.