Enhanced Primary Human Liver Sinusoidal Endothelial Cells are expanded primary cells that retain the properties of primary liver sinusoidal endothelial cells which allows for a reliable in vitro system. These cells are ready-to-use for applications in research and development, such as viral infections, screening, co-cultures, transient transfection, and for toxicity studies. The cells offer a unique ability to be kept in longer term culture when compared to standard primary liver sinusoidal endothelial cells which does not proliferate well in vitro.The cells are offered ready-to-use after thawing. Recommend to only passage the cells 1-2 more times, as more passages may see phenotypical changes and expression of senescence genes.
Cell Type
Primary Cells
Disclaimer
Enhanced Primary Human Liver Sinusoidal Endothelial Cells are expanded primary cells that retain the properties of primary liver sinusoidal endothelial cells which allows for a reliable in vitro system. These cells are ready-to-use for applications in research and development, such as viral infections, screening, co-cultures, transient transfection, and for toxicity studies. The cells offer a unique ability to be kept in longer term culture when compared to standard primary liver sinusoidal endothelial cells which does not proliferate well in vitro.The cells are offered ready-to-use after thawing. Recommend to only passage the cells 1-2 more times, as more passages may see phenotypical changes and expression of senescence genes.
Gender
Donor Info Not Disclosed
Growth Properties
Adherent
Quantity
5x105 cells / 1.0 ml
Tissue
Liver
Morphology
Endothelial-like
Species
Human (H. sapiens)
Propagation
Use of PriCoatTM T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.Use the ready-to-use Human Liver Sinusoidal Endothelial Cell Thawing Medium (TM111) and the Human Liver Sinusoidal Endothelial Cell Media Kit (TM112) which comes with the Human Liver Sinusoidal Endothelial Cell Basal Medium, and Human Liver Sinusoidal Endothelial Cell Supplement Mix available from abm.Carbon dioxide (CO2): 5%, Temperature: 37.0°C.To make complete Human Liver Sinusoidal Endothelial Cell Growth Medium add the entire content of Human Liver Sinusoidal Endothelial Cell Supplement Mix into Human Liver Sinusoidal Endothelial Cell Basal Medium and mix properly in BioSafety Cabinet. Addition of the Human Liver Sinusoidal Endothelial Cell Supplement Mix may make medium appear more opaque.To Thaw:1. Pre-warm Human Liver Sinusoidal Endothelial Cell Thawing Medium and fully supplemented Human Liver Sinusoidal Endothelial Cell Growth Medium to room temperature.2. Carefully remove cryovial from storage tank.3. Thaw cells in 37°C water bath until only a small chunk of ice is left. Do not shake the vial, or take it out of the water during thawing, as this will damage the cells.4. Spray the vial and the tube containing 50 ml of thawing medium with 70% ethanol and transfer to a Biosafety cabinet.5. Transfer the now completely thawed cell suspension from the cryovial into 10 ml Human Liver Sinusoidal Endothelial Cell Thawing Medium in a new 50 ml conical tube by gently pipetting the cells into the medium using a 2 ml pipette.6. Use a 1 ml pipette to transfer 1 ml of the thawing medium with the cells back to the cryovial and pipette the contents back into the 50 ml tube. Repeat this process twice to completely remove the cells from the cryovial.7. Pellet the cells by centrifuging at 620×g for 5 min at RT. Important note: Higher g-forces will significantly reduce cell recovery.8. Aspirate the supernatant without disturbing the pellet. Leave approximately 200 mul medium on top of the cells.9. Add 800 mul of the fully supplemented Human Liver Sinusoidal Endothelial Cell Growth Medium and gently loosen and re-suspend the cells by pipetting them up and down 1-2 times. Do not vortex or shake the cells as this will compromise cell survival.11. Determine viable cell number by cell count.12. Dilute the Enhanced Primary Human Liver Sinusoidal Endothelial Cells in pre-warmed, fully supplemented Human Liver Sinusoidal Endothelial Cell Growth Medium and seed at ~10,000 cells/cm2 in ECM-coated cell culture flasks (G422) or appropriate cell culture dishes.13. Incubate the cells at 95% humidity, 37°C and 5% CO2.14. Next day, add fresh new Human Liver Sinusoidal Endothelial Cell Growth Medium. Conduct complete media change every other day with fresh Human Liver Sinusoidal Endothelial Cell Growth Medium for optimal growth.
Applications
For Research Use Only
Subculture Protocol
To Subculture:1. Pre-warm PBS, trypsin/EDTA and Human Liver Sinusoidal Endothelial Cell Growth Medium to 37°C.2. Carefully aspirate the culture supernatant.3. Wash the plate once with ~100 µl PBS/cm2.4. Add ~20 µl/cm2 trypsin/EDTA (0.05%/0.02% EDTA).5. Incubate for 3-5 min at 37°C until most of the cells are rounded up. Check under microscope. Avoid incubating the cells for more than 10 minutes.6. Gently tap the cell culture vessel to detach the rounded up cells.7. Add pre-warmed Human Liver Sinusoidal Endothelial Cell Growth Medium at equal volume of the trypsin used. Rinse the remaining attached cells from the culture surface using a pipette.8. Transfer the complete suspension to a tube and centrifuge at 620×g for 5 min at RT.9. Aspirate the supernatant without disturbing the pellet. Add pre-warmed Human Liver Sinusoidal Endothelial Cell Growth Medium (~2-3 ml).10. Gently re-suspend the cells with pipette. Do not vortex or shake the cells as this will compromise cell survival.11. Determine viable cell number by cell count.12. Dilute the Enhanced Primary Human Liver Sinusoidal Endothelial Cells in pre-warmed, fully supplemented Human Liver Sinusoidal Endothelial Cell Growth Medium and seed at ~10,000 cells/cm2 in ECM-coated cell culture flasks (G422) or appropriate cell culture dishes.13. Incubate the cells at 95% humidity, 37°C and 5% CO2.
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