Table 1.

Overview of EHM protocols.

	Component	Starting Protocol	Matrix Protocol	Serum-free Protocol
EHM reconstitution medium	Collagen rat (research grade)	0.4 mg/EHM
	Collagen bovine (medical grade)		0.4 mg/EHM	0.4 mg/EHM
	Matrigel®	10% v/v
	Reconstitution Medium	DMEM	DMEM	RPMI
	Horse Serum	10%
	Chick Embryo Extract	2%
	Fetal Bovine Serum		40%
	B27 (without insulin)			4%
EHM culture medium	Base Medium	Iscove	Iscove	Iscove*
	Fetal Bovine Serum	20%	20%
	B27 (without insulin)			4%
	IGF-1			100 ng/mL
	FGF-2			10 ng/mL
	VEGF165			5 ng/mL
	TGF-β1			5 ng/mL
	Non-Essential Amino Acids	1%	1%	1%
	Glutamine	2 mmol/L	2 mmol/L	2 mmol/L
	Penicillin	100 U/mL	100 U/mL	100 U/mL
	Streptomycin	100 μg/mL	100 μg/mL	100 μg/mL
	β-Mercaptoethanol	100 μmol/L	100 μmol/L

^*Alternatively other basal medium with ≥1.2 mmol/L calcium