All hiPSC lines used in this study have were derived as previously described11 (link) under Addenbrooke’s Hospital Ethics reference number 08/H0311/201; R&D No. A091485. These cells were maintained at 37 °C in humidified incubators supplemented with 5% v/v carbon dioxide and were differentiated using our established hepatic differentiation protocol with minor modifications:14 (link) Cells were differentiated at 5% oxygen and two splitting steps allowed for scale-up and prolonged maturation of differentiating cells (Fig. S1A ). During the splitting steps on day 8 and day 25, cells were washed with PBS and dissociated with TrypLE™ (Life Technologies, 12563–029) for 20–45 min at 37 ° C. Harvested cells were centrifuged at 100 × g for 3 min. Cells were resuspended in RPMI (day 8 split) or HepatoZYME-SFM (day 25) (Thermo Fisher, 17705–021). Cells were counted and resuspended in RPMI and Activin (day 8 split) or HepatoZYME-SFM (Thermo Fisher, 17705–021), OSM and HGF (day 25 split), supplemented in each case with ROCK inhibitor (1 μL/ml) to a concentration of 210,500 cells/cm2.
Hepatozyme sfm
Manufactured by Thermo Fisher Scientific
Sourced in United States
HepatoZYME-SFM is a serum-free, protein-free medium designed for the culture of primary hepatocytes and hepatoma cell lines. It provides the necessary nutrients and growth factors to support the growth and maintenance of hepatocyte cultures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Liver perfusion medium, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Liver digest medium, by Thermo Fisher Scientific (2 mentions)
Growth factor, by Thermo Fisher Scientific (2 mentions)
A83 01, by Thermo Fisher Scientific (2 mentions)
Dihexa, by Thermo Fisher Scientific (2 mentions)
Human insulin, by Merck Group (1 mentions)
High glucose dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Williams e medium, by Merck Group (1 mentions)
Gw9662, by Merck Group (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Troglitazone, by Merck Group (1 mentions)
Liberase blendzyme 3, by Roche (1 mentions)
70 μm nylon mesh filter, by BD (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Human oncostatin m, by Thermo Fisher Scientific (1 mentions)
20 protocols using hepatozyme sfm
1
Scalable hiPSC-derived Hepatic Differentiation
2
Isolation and Culture of Primary Murine Hepatocytes and HepG2 Cells
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Primary murine HPC were isolated from male C57BL/6 mice according to the collagenase method of Seglen
[50 (link)]. Cells were plated in collagen coated 6-well dishes at a density of 1.2 × 106 cells using HepatoZYME-SFM (Gibco, Life Technology, Darmstadt, Germany). Four hours after seeding the medium was renewed and cells were grown for a further 24 hrs culture period.
HepG2 (DSMZ: DSM ACC180) were cultured in RPMI (PAA, Pasching, Austria) containing 10% fetal calf serum (PAA), 1 × Penicillin/Streptomycin (Lonza, Cologne, Germany). Medium was renewed every second day. For the experiment, cells were passaged and plated in 6-well dishes using accutase (PAA) at a density of 4 × 105 cells. One day before the experiment, cells were washed with PBS (1×), medium changed to HepatoZYME-SFM (Gibco) and cultured for further 24 hrs.
[50 (link)]. Cells were plated in collagen coated 6-well dishes at a density of 1.2 × 106 cells using HepatoZYME-SFM (Gibco, Life Technology, Darmstadt, Germany). Four hours after seeding the medium was renewed and cells were grown for a further 24 hrs culture period.
HepG2 (DSMZ: DSM ACC180) were cultured in RPMI (PAA, Pasching, Austria) containing 10% fetal calf serum (PAA), 1 × Penicillin/Streptomycin (Lonza, Cologne, Germany). Medium was renewed every second day. For the experiment, cells were passaged and plated in 6-well dishes using accutase (PAA) at a density of 4 × 105 cells. One day before the experiment, cells were washed with PBS (1×), medium changed to HepatoZYME-SFM (Gibco) and cultured for further 24 hrs.
3
Hepatocyte Cell Culture Protocol
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GW9662, troglitazone, ethylenediaminetetraacetic acid, dexamethasone, collagenase IV, Williams’ Medium E, porcine GH, human insulin, and 20 kinds of AAs were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was purchased from LC Laboratories (Massachusetts, USA). Penicillin–streptomycin, fetal bovine serum, high glucose Dulbecco's modified Eagle’s medium (DMEM), and hepatozyme-SFM were obtained from Life Technologies (Invitrogen, Carlsbad, CA, USA). The AA-free medium was procured from Jiang Lai Bio-Technology Co., Ltd. (Shanghai, China). Human GH was obtained from Abaier Bio-Technology Co., Ltd. (Shenzhen, China). HepG2 cells (ATCC) were purchased from Beijing zhongyuan Co., Ltd. (Beijing, China).
4
Tissue Dissociation and SP Isolation
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Tissue dissociation and SP isolation were done as described previously.8 (link) The samples stored in liquid nitrogen were thawed at 37 °C and washed with Hank’s balanced salt solution (Life Technologies, UK). After dissociation using Liberase Blendzyme 3 (Roche, Basel, Switzerland) at a concentration of 0.8 Wunsch unit/ml during 1 h at 37 °C, the samples were filtered with a 70 μm nylon mesh filter (BD Biosciences, Erembodegem, Belgium) and gradient centrifugation based on 15% Percoll/Hank’s balanced salt solution (100 × g for 15 min). Finally, the pelleted cells were resuspended in HepatoZYME-SFM (Life Technologies, UK) with 1% penicillin/streptomycin (Life Technologies, UK) added to obtain a single-cell suspension.
5
Hepatic Differentiation Protocol
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RPMI, 50× B27 Supplement, Knockout DMEM (KO-DMEM), Knockout Serum Replacement Medium (KO-SR), GlutaMAX, Penicillin/Streptomycin (P/S), and HepatoZYME-SFM (HZM) were purchased from Life Technologies. Recombinant Mouse Wnt3a, Human Activin A (AA), Human Hepatocyte Growth Factor (HGF), and Human Oncostatin M (OSM) were from PeproTech (Hannoun et al., 2010; Hay et al., 2011; Szkolnicka et al., 2013 ).
6
Isolation of Primary Mouse Hepatocytes
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Primary hepatocytes were isolated from 8- to 12-week-old male C57BL/6J mouse51 (link). Briefly, C57BL/6J mouse was anaesthetized and perfused with 20 mL calcium-free buffer from the inferior vena cava to the liver, and then the buffer containing collagenase I (LS004196, Worthington, USA) was added to breakdown the extracellular matrix and tight junctions at 37 °C. The liver was dissected in a sterile 6-cm cell culture dish containing 10 mL cold DMEM with 10% FBS, followed by filtration through a 70 μm cell strainer and centrifugation at 500 rpm for 3 min. Cells were then centrifuged with Percoll (P1644-100 ML, Sigma–Aldrich, USA) and living cells were resuspended in Hepato ZYME-SFM (17705-021, Thermo Fisher, USA) medium supplemented with 2 mmol/L l -glutamine, 20 units/mL penicillin and 20 μg/mL streptomycin. Then the hepatocytes were plated at 6 × 105 cells/well in 6-well or 1.5 × 105 cells/well in 24-well culture dishes precoated with 0.2% glue.
7
Isolation of Primary Mouse Hepatocytes
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Hepatocytes were isolated from the livers of the mice, as described in the manufacturer's instructions. Briefly, after the mice were anesthetized, the inferior vena cava (IVC) and portal vein were exposed. The IVC was cannulated with a 24G soft needle and perfused with Liver Perfusion Medium (Gibco) at 4 mL/min for 5 min. Next, the perfusion medium was replaced with liver digestion medium (Gibco) at 3.5 mL/min for 10 min. The liver was collected into a dish with DMEM, and the liver sac was cut to release hepatocytes. The liver cell-suspension was filtered through a 70-μm mesh filter and spun at 100×g for 5 min. The pellet was re-suspended in attachment medium and added to collagen I-coated dishes (Matsunami, Osaka, Japan). After cell attachment (approximately 3 h after plating), primary hepatocytes were cultured at a density of 1.0 × 105 cells/well in HepatoZYME-SFM (Thermo-Fisher) containing 1 × penicillin-streptomycin, 2 mM l -glutamine and 1.25 μg/cm2 type I collagen, at 37 °C in a humidified atmosphere containing 5% CO2.
8
Evaluating Culture Media for Human iPS-HLCs and PHH
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Human iPS-HLCs and PHH were cultured with following five culture mediums.
Hepatocyte Culture Medium Bullet Kit™ (Lonza, HCM);
HepatoZYME-SFM (Thermo Fisher Scientific, HepatoZYME);
Cellartis Power Primary HEP Medium (Cellartis, Primary HEP);
DMEM/F12 (Thermo Fisher Scientific) supplemented with Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, DMEM/F12);
William’s E Medium, no phenol red (Thermo Fisher Scientific) supplemented with Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, WEM).
Hepatocyte Culture Medium Bullet Kit™ (Lonza, HCM);
HepatoZYME-SFM (Thermo Fisher Scientific, HepatoZYME);
Cellartis Power Primary HEP Medium (Cellartis, Primary HEP);
DMEM/F12 (Thermo Fisher Scientific) supplemented with Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, DMEM/F12);
William’s E Medium, no phenol red (Thermo Fisher Scientific) supplemented with Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, WEM).
9
Mitochondrial Isolation and Respiration Assay
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Rotenone, ADP, antimycin A, L-glutamic acid, L-malic acid, succinate disodium, fatty acid–free BSA, collagenase type IV, Percoll®, and glucose were purchase from Millipore Sigma Corp. Calcium ionophore A23187, N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, nordihydroguaiaretic acid (NDGA), ibuprofen, oligomycin, cell culture supplements, thromboxane B2-d4, prostaglandin E2-d4, 12-HETE-d8, and 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) were purchased from Cayman Chemical Company. The lactate dehydrogenase (LDH)-cytotoxicity assay kit, HepatoZYME-SFM, and other chemicals for preparation of buffers for mitochondrial isolation, respiration, and swelling were obtained from ThermoFisher Scientific Inc. A rabbit polyclonal anti-iPLA2γ antibody was generated in our laboratory as described previously (15 (link)).
10
Hepatocyte Differentiation from hPSCs
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hPSCs were dissociated into single cells following incubation with StemPro Accutase (Thermo Fisher) for 5 min at 37°C and seeded at a density of 50,000 cells/cm2 in E8 medium supplemented with 10 µM ROCK Inhibitor Y-27632 (Selleckchem). Hepatocytes were differentiated 48 hr after seeding, as previously reported (Hannan et al., 2013 (link)) with minor modifications. Following endoderm differentiation, anterior foregut specification was achieved with RPMI-B27 differentiation media supplemented with 50 ng/ml Activin A (R&D) for 5 days. Cells at the foregut stage were further differentiated into hepatocytes with Hepatozyme complete medium: HepatoZYME-SFM (Thermo Fisher) supplemented with 2 mM L-glutamine (Thermo Fisher), 1% penicillin-streptomycin (Thermo Fisher), 2% non-essential amino acids (Thermo Fisher), 2% chemically defined lipids (Thermo Fisher), 14 μg/ml of insulin (Roche), 30 μg/ml of transferrin (Roche), 50 ng/ml hepatocyte growth factor (R&D), and 20 ng/ml oncostatin M (R&D), for up to 27 days.
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