Human recombinant epidermal growth factor

HLSCs were obtained and characterized as previously described (Herrera et al., 2006 (link); Bruno et al., 2019 (link); Spada et al., 2020 (link)). HLSCs were expanded in the presence of Minimal Essential Medium (α-MEM, Lonza, Basel, Switzerland) supplemented with 10% Fetal Calf Serum (Gibco/Cambrex, Invitrogen, Carlsbad, CA, United States), 10 ng/mL of human recombinant Epidermal Growth Factor, 10 ng/mL of human recombinant Fibroblast Growth Factor basic (Miltenyi Biotec, Bergisch Gladbach, Germany) and used until passage 8 (Bruno et al., 2019 (link); Spada et al., 2020 (link)). Human bone marrow-derived mesenchymal stromal cells (MSCs) were purchased by Lonza (Basel, Switzerland), maintained in a MSC basal medium bullet kit (Lonza) and used until passage 6. Human hepatic stellate cells LX-2 (Xu et al., 2005 (link)) (Sigma Aldrich, St. Louis, MO, United States), were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (4.5 g/L, Euroclone, Pero, MI, Italy) supplemented with 2% Fetal Calf Serum and 2 nM L-Glutamine (Lonza) and used until passage 8. All cells were maintained in a humidified 5% CO2 incubator at 37°C.

HLSCs were obtained and cultured as previously described.³ (link)^,4 (link) Briefly, HLSCs were generated by Anemocyte International (Gerenzano, Italy) from a 10- to15-mm liver fragment obtained from a liver donor, according to the standard criteria of Centro Nazionale Trapianti, as described previously.³ (link)^,4 (link) The liver fragment was enzymatically digested with good manufacturing practice (GMP)-grade collagenase (NB 1, 0.6 mg/mL) and neutral protease (NBI, 0.73 mg/mL) for 30 min at 37°C. The liver cell suspension was washed (400 × g for 10 min) and cultured (2.5 × 10⁵/mL in a T75 flask with 10 mL per flask) in the presence of minimal essential medium (α-MEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (GIBCO, Cambrex), 10 ng/mL human recombinant epidermal growth factor (Miltenyi, Bergisch Gladbach, Germany), 10 ng/mL human recombinant basic fibroblast growth factor (Miltenyi, Bergisch Gladbach, Germany), 2 nM L-glutamine (Lonza), and 100 U/mL penicillin/streptomycin (Sigma, St. Louis, MO, USA) and maintained in a humidified 5% CO₂ incubator at 37°C. After 2 weeks of culture, cells were seeded at a density of 2.5 × 10⁵ cells per flask (T75) in the same culture medium for expansion.

HLSCs were isolated and maintained in a culture as previously described [34 (link),47 (link),48 (link)]. HLSCs were plated at a density of 2.5 × 10⁵ cells per flask (T75, Corning, VWR International, Milano, Italy) and maintained in the presence of Minimal Essential Medium (α-MEM, Lonza, Basel, Switzerland) with 10% Fetal Calf Serum (Gibco/Cambrex, Invitrogen, Carlsbad, CA, USA), specific growth factors (10 ng/mL of human recombinant Epidermal Growth Factor and of human recombinant Fibroblast Growth Factor basic, Miltenyi, Bergisch Gladbach, Germany), 2nM L-Glutamine (Lonza), and antibiotics (100U/mL of Penicillin/Streptomycin, Sigma-Aldrich, St. Louis, MO, USA).