Adult liver progenitor cells are isolated from healthy human livers by enzymatic digestion and expanded in culture.
Liver segments are sequentially perfused with EDTA solution (Earle's balanced salt solution without Ca++. Mg++, 0.5 mM EDTA) followed by collagenase solution [Earle's balanced salt solution with Ca++, Mg++, 180 mg/400 mL collagenase B, 8 mg/400 mL soybean trypsin inhibitor] for 15 min each, at 37°C. Hepatocytes are physically separated by gentle shaking in the collagenase solution, filtered through sterile gauze, washed, and plated on Primaria plates at densities of 2 X 104/cm2 in medium with serum (10%). After 8-24 h, medium is replaced with a hormonally-defined medium (SUM-Chow-dhury 3: 1, minimum essential medium:Waymouths (tyrosine-free), insulin 1.7 x 10-8 M (100 ng/mL), glucagon, 2.8 x 10-7 M (1 μg/mL), epidermal growth factor 8.3 x 10-9 M (50 ng/mL), somatotropin 9.5 x 10-11 M (10 μU/mL), linoleic acid 1.8 x 10-5 M (5 μg/mL), selenium 3 nM, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4) 10 mM, dexamethasone 10-7 M, thyroxine 1 μM, transferrin 1.3 x 10-7 M (10 μg/mL), Gly-His-Lys 6.5 x 10-8 M (20 ng/mL)).