Mouse primary hepatocytes were isolated from 8- to 10-week-old male livers using a two-step collagenase perfusion method [13] (link). In 3.5-cm Petri dishes coated with fibronectin (0.5 mg/ml, Sigma), 5 × 105 cells were seeded in Williams’ medium E (Invitrogen) with 10% FBS. After 2 h, the medium was replaced with hepatozyme medium (Invitrogen) for 12 h prior to transfection. We first generated cDNAs of human and mouse Apolipoprotein F (ApoF) tagged with V5 at their C-termini. These were co-transfected in mouse primary hepatocytes with cDNAs coding for either an empty vector control, WT PC7 or its R504H mutant. Transfections were performed with Effectene using a total of 4 μg of cDNA, following the manufacturer’s instructions. Cell lysates and media were collected 48 h post-transfection and subjected to SDS–PAGE separation (14% Tris-Tricine) followed by Western blot analysis.
Hepatozyme medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hepatozyme medium is a specialized cell culture medium designed to support the growth and maintenance of hepatocytes, which are the principal functional cells of the liver. It provides the necessary nutrients and growth factors to facilitate the in vitro culture of hepatocytes derived from various sources. The medium is formulated to maintain the specific metabolic and functional characteristics of hepatocytes in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (4 mentions)
William s e medium, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Collagenase type 5, by Merck Group (2 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (2 mentions)
Oncostatin m, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (1 mentions)
Fumed silica, by Merck Group (1 mentions)
Primaria dishes, by BD (1 mentions)
35s cys met, by MP Biomedicals (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Collagen coated 24 well plates, by Greiner (1 mentions)
Chemical defined lipid concentrate, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Dxc 600 beckman, by Beckman Coulter (1 mentions)
Luciferin pfbe substrate, by Promega (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
11 protocols using hepatozyme medium
1
Mouse Hepatocyte Transfection Assay
2
Hepatocyte Lipid Metabolism Analysis
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Dulbecco's modified Eagle's medium (DMEM), Met/Cys-free DMEM, Williams Medium E, Hepatozyme medium, fetal bovine serum (FBS), and antibiotic/antimycotic were purchased from Invitrogen Canada (Burlington, ON). Heparin, fibronectin, and fumed silica were purchased from Sigma-Aldrich (Oakville, ON). The Primaria dishes were from BD Biosciences (Mississauga, ON), and the [35S]Met/Cys was obtained from MP Biochemicals (Solon, OH). Protease inhibitor cocktail and chemiluminescent substrates were purchased from Roche Diagnostics (Laval, PQ). The antibody to detect hHL (by immunoblot analysis) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), the antibodies to detect mouse apoE and apoA-I (for immunoblot and immunoprecipitation) were purchased from BioDesign International (Saco, ME, USA), and the antibodies to detect mouse calnexin and giantin were obtained from Stressgen Bioreagents (Ann Arbor, MI, USA) and Abcam (Cambridge, MA, USA), respectively. The anti-hHL antibody used for immunoprecipitation of HL was a kind gift from Dr. Ann White (University of Texas Southwestern Medical Center).
3
Cultivation of iPSC-Derived Human Hepatocytes
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The iPSC derived human hepatocytes (Definigen, Cambridge, England) were obtained as a cryopreserved sample and cells were recovered for 12 days following the manufacturer’s instructions. Briefly, cells were thawed on collagen-coated 24 well plates (Greiner Bio-One, Frickenhausen, Germany) at density of 5 × 105 cells per well using minimum essential medium, supplemented with 2% non-essential amino acids, 2% chemical defined lipid concentrate (Thermo Fisher Scientific, Waltham, MA), 0.1% insulin (Sigma, Saint Louis, MO) and manufacturer’s cytokines. Cells were cultured in Hepatozyme medium (Invitrogen, Waltham, MA), supplemented with 2% non-essential amino acids, 2% chemical defined lipid concentrate (Thermo Fisher Scientific, Waltham, MA), 0.1% insulin (Sigma, Saint Louis, MO) and manufacturer’s cytokines, and were maintained at 37 °C with 20% O2.
4
Isolation of Hepatocytes from Mice Livers
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Hepatocytes were isolated from 12-weeks-old male mice livers using the two-step collagenase perfusion method. After anesthesia of mice by 2% isoflurane inhalation, the peritoneal cavity was opened, and the liver was perfused in situ via the inferior vena cava for 6 min at 37 °C with calcium-free HEPES buffer I (142 mm NaCl, 6.7 mm KCl, 10 mm HEPES, pH 7.6) and for 8 min with calcium-supplemented HEPES buffer II (4.7 mm CaCl2, 66.7 mm NaCl, 6.7 mm KCl, 100 mm HEPES, pH 7.4) containing 0.5 mg/ml collagenase type V (Sigma Aldrich). The perfusion rates were set to 8 and 6 ml/min, respectively. In 3.5-cm Petri dishes coated with fibronectin (0.5 mg/ml, Sigma Aldrich), 0.5x10 6 cells were seeded in Williams' medium E supplemented with 10% fetal bovine serum (Invitrogen). After 2 h, the medium was replaced with hepatozyme medium (Invitrogen) for 12 h before the treatment.
5
Organoid-Based Hepatic Functional Characterization
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For determination of albumin secretion, a culture medium from differentiated organoids was collected at day 14 of differentiation. Protein in the medium was concentrated using Amicon Ultra centrifugal filters (Millipore), and the amount of albumin was measured using a DxC-600 Beckman (Beckman Coulter). The values were normalized for total cell number. For measurement of cytochrome P450 activity, 14-day-differentiated organoids were removed from Matrigel and incubated in 50 μM luciferin-PFBE substrate (Promega) in hepatozyme medium (Gibco) containing 10% FBS for 8 hr at 37°C. Cyp3a activity was then measured with a luminometer using the P450-Glo cytochrome P450 assay kit according to the manufacturer’s instructions (Promega). For comparative analysis, freshly isolated canine hepatocytes were used (Arends et al., 2009 (link)). Furthermore, human cell lines HepG2 (cultured in DMEM with 10% FBS) and Huh 7 (cultured in DMEM with 10% FBS) were included for comparative functional analysis.
For measurement of the expression of hepatic enzymes in differentiated organoids, cells were lysed in milliQ at day 14 and stored at −20°C. ALT and AST were measured using the DxC-600 Beckman (Beckman Coulter) standard protocols, and values were corrected for total cell counts.
For measurement of the expression of hepatic enzymes in differentiated organoids, cells were lysed in milliQ at day 14 and stored at −20°C. ALT and AST were measured using the DxC-600 Beckman (Beckman Coulter) standard protocols, and values were corrected for total cell counts.
6
Isolation of Primary Hepatocytes from Pcsk9-/- Mice
7
Isolation and Culture of Macrophages, Hepatocytes
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Thioglycollate-elicited macrophages and HepG2 cells were maintained in RPMI-1640 with 10% FBS and 100 U/mL penicillin/streptomycin (Cellgro/Corning). Primary mouse hepatocytes were prepared as described and maintained in HepatoZYME medium (Gibco) with 10% FBS, 1% l -glutamine, and 1% penicillin/streptomycin (refer to Supporting Information for additional details).
8
Generation of iPSC-derived Liver Organoids
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iPSC-derived hepatic progenitors (IH) were generated using our established protocol (Fig. 1 B) [13 (link)]. To generate iPSC liver organoids, we suspended approximately 0.5 × 106 IH cells in Hepatozyme medium (Life Technologies), supplemented with Oncostatin M 0.01 μg/ml (Peprotech) and Hepatocyte Growth Factor 0.05 μg/ml (Peprotech) with the final cell density of 125 × 106 cells/ml (Fig. 1 C). Approximately 4 μl cell suspension was then pipetted onto top and bottom surfaces of partially dehydrated ICC scaffolds and placed in an incubator for 30 min without media to minimize cell loss and maximize cell attachment. Cell-laden ICC scaffolds were then transferred to a new 96-well plate for culture and media were refreshed at 4 h post-seeding to remove excessive cells that are not attached to surface or established cellular clusters. Cells were left for a further 14 days to mature. Media was refreshed every two days. 2D controls were generated using IH cells seeded into standard 48 well plate tissue culture plastic plates coated with the same ECM proteins and cultured with the same reagents as used for ICC culture.
9
Isolation of Mouse Hepatocytes
10
Metabolic Profiling of PEX-Treated Hepatocytes
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PHH (Xenotech, Kansas City, KS,
20 donors, Cat No. HPCH20-50, Batch No. 1910146) were thawed and plated
in 12-well plates (Corning, Corning, NY) according to the protocol
of the vendor with the density of 6.95 × 105 cells/well.
The cells were cultured in complete HepatoZYME medium, containing
GlutaMAX, Insulin-Transferrin-Selenium, and penicillin/streptomycin
(all of the reagents were obtained from Thermo, San Jose, CA) at 37
°C in a humidified atmosphere with 5% CO2 for 24 h
before PEX treatment. The PHH were treated with 20 μM PEX with
or without 10 μM KCZ for 6 h. The medium was then transferred
into Eppendorf tubes and centrifuged at 100 rcf for 5 min to remove
the suspending cells. The cells were washed with 1× DPBS (Thermo,
San Jose, CA) 3 times, harvested in 500 μL of methanol–water
(v/v 1/1), and lysed with a probe ultrasonicator (Thermo, San Jose,
CA). Twenty microliters of culture medium was added to 60 μL
of ice-cold acetonitrile containing 0.1 μM agomelatine as the
internal standard (IS), or 50 μL of cell lysate was added with
100 μL of IS solution. After vortexing and centrifugation at
15,000 rcf for 15 min, the supernatants were transferred to sample
vials and 3 μL was injected into a UHPLC-Q Exactive MS system
for analysis.
20 donors, Cat No. HPCH20-50, Batch No. 1910146) were thawed and plated
in 12-well plates (Corning, Corning, NY) according to the protocol
of the vendor with the density of 6.95 × 105 cells/well.
The cells were cultured in complete HepatoZYME medium, containing
GlutaMAX, Insulin-Transferrin-Selenium, and penicillin/streptomycin
(all of the reagents were obtained from Thermo, San Jose, CA) at 37
°C in a humidified atmosphere with 5% CO2 for 24 h
before PEX treatment. The PHH were treated with 20 μM PEX with
or without 10 μM KCZ for 6 h. The medium was then transferred
into Eppendorf tubes and centrifuged at 100 rcf for 5 min to remove
the suspending cells. The cells were washed with 1× DPBS (Thermo,
San Jose, CA) 3 times, harvested in 500 μL of methanol–water
(v/v 1/1), and lysed with a probe ultrasonicator (Thermo, San Jose,
CA). Twenty microliters of culture medium was added to 60 μL
of ice-cold acetonitrile containing 0.1 μM agomelatine as the
internal standard (IS), or 50 μL of cell lysate was added with
100 μL of IS solution. After vortexing and centrifugation at
15,000 rcf for 15 min, the supernatants were transferred to sample
vials and 3 μL was injected into a UHPLC-Q Exactive MS system
for analysis.
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