Recipients for human hepatocytes were produced by crossbreeding POR cKO mice and TK-NOG mice, a hepatic injury model. The methods for liver humanization employed in the TK-NOG model have been described previously22 (link). Briefly, the mice were given 0.06 mg/mL valganciclovir for 3 days to ablate hepatocytes expressing the HSV-TK transgene. Human hepatocytes (1.0 × 106 cells/mouse) were injected into the spleens of liver-injured mice. Cryopreserved human hepatocytes (12-year-old Caucasian female [donor A] and 30-year-old African American female [donor B]) were provided by Lonza Walkersville Inc. (Walkersville, MD, USA). The replacement index of the humanized liver was evaluated by measuring the blood levels of human albumin with a human albumin ELISA quantitation kit (Bethyl Laboratories, Montgomery, TX, USA) or butyrylcholinesterase (ChE) activity (Fuji Dri-Chem 7000; Fuji Photo Film, Tokyo, Japan).
Cryopreserved human hepatocytes
Manufactured by Lonza
Sourced in United States, Switzerland
Cryopreserved human hepatocytes are primary liver cells derived from human donors and preserved in a cryogenic state. They are used for in vitro research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Human albumin elisa quantitation kit, by Fortis Life Sciences (1 mentions)
Fuji dri chem 7000, by Fujifilm (1 mentions)
Cryopreserved hepatocyte recovery medium, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Merck Group (1 mentions)
Trypan blue, by Lonza (1 mentions)
Diclofenac, by LGC (1 mentions)
Anamorelin, by LGC (1 mentions)
3 protocols using cryopreserved human hepatocytes
1
Humanized Liver Mouse Model
2
Cryopreserved Human Hepatocytes Protocol
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Cryopreserved human hepatocytes (Lonza) were used
in this study.
The vials of PHHs were rapidly thawed in a shaking water bath at 37
°C, the contents of each vial were emptied in pre-warmed cryopreserved
hepatocyte recovery medium (Thermo Fisher Scientific), and the suspension
was centrifuged at 1200 rpm for 5 min at room temperature. The PHH
was suspended at 5 × 106 cells/mL in HCM (Lonza) containing
10% fetal calf serum (FCS). For the PS plates, the PHH was seeded
at 1.0 × 105 cells/cm2 in HCM (Lonza) containing
10% FCS onto type I collagen-coated 96-well plates. For the PDMS devices,
the devices were coated with type I collagen before 10 μL of
cell suspension was injected. For the FEPM devices, 20 μL of
cell suspension was injected without type I collagen coating. After
1 h, 200 μL of medium was added into each of the top and bottom
channels of the devices. PHHs, which were cultured 24 h after the
plating, were used in the experiments.
in this study.
The vials of PHHs were rapidly thawed in a shaking water bath at 37
°C, the contents of each vial were emptied in pre-warmed cryopreserved
hepatocyte recovery medium (Thermo Fisher Scientific), and the suspension
was centrifuged at 1200 rpm for 5 min at room temperature. The PHH
was suspended at 5 × 106 cells/mL in HCM (Lonza) containing
10% fetal calf serum (FCS). For the PS plates, the PHH was seeded
at 1.0 × 105 cells/cm2 in HCM (Lonza) containing
10% FCS onto type I collagen-coated 96-well plates. For the PDMS devices,
the devices were coated with type I collagen before 10 μL of
cell suspension was injected. For the FEPM devices, 20 μL of
cell suspension was injected without type I collagen coating. After
1 h, 200 μL of medium was added into each of the top and bottom
channels of the devices. PHHs, which were cultured 24 h after the
plating, were used in the experiments.
3
Bioanalysis of Anamorelin and Diclofenac
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We procured pure standards of anamorelin and diclofenac (experimental control) from Toronto Research Chemicals (Toronto, ON, Canada) and Sigma-Aldrich (Milan, Italy), respectively. Stock solutions of the standards were prepared at 1 mg/mL in methanol and stored at −20 °C. LC-MS grade acetonitrile, water, methanol, and formic acid were obtained from Carlo Erba (Cornaredo, Italy). Cryopreserved human hepatocytes (pooled from ten donors), thawing medium, and trypan blue (0.4%) were obtained from Lonza (Basel, Switzerland). Williams’ medium E (WME), l-glutamine, and HEPES buffer (2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid), were also procured from Sigma-Aldrich. HEPES (20 mmol/L) and l-glutamine (2 mmol/L) were used in preparing Supplemented Williams’ Medium E (SWM) and stored at 4 °C until incubation.
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