Enhanced Primary Human Liver Sinusoidal Endothelial Cells

Cat. No.

T5997

Unit

5x10⁵ cells / 1.0 ml

Price

Inquiry

Specifications

Description	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. To Thaw:
Cat. No.	T5997
Name	Enhanced Primary Human Liver Sinusoidal Endothelial Cells
Unit	5x10⁵ cells / 1.0 ml
Price	Inquiry
Category	Primary Cells
Cell Type	Primary Cells
Organism	Human (H. sapiens)
Tissue	Liver
Cell Morphology	Endothelial-like
Growth Properties	Adherent, endothelial-like
Seeding Density (cells/cm²)	12,500
Propagation Method	Use of PriCoat^TM 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 μl medium on top of the cells. 9. Add 800 μl 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.
Growth Conditions	Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. 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 are required for growth of the cells, 37.0°C, 5% CO₂.
Subculture Protocol	Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 80% confluent. 1. Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel. 2. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment. 3. Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel. 4. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type. 5. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired. 6. Incubate the cells at the recommended conditions.
QC	Each lot has been tested for: >90% plateability Post-thaw viability CMV, HIV, HBV, HCV, and Mycoplasma absence Liver sinusoidal endothelial cell markers via immunofluorescence staining: MMR+ and CD31+
Application	Research Use Only.
Storage Condition	Vapor phase of liquid nitrogen, or below -130°C.
BioSafety	II
Disclaimer	1. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic or any other non-RUO application(s). 2. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature are provided for informational purposes only. abm does not warrant that such information has been shown to be accurate. 3. abm warrants that cells shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period".

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FAQs

	I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
	We do not perform extensive downstream characterization, or gene expression profiling of our cell lines. However, you can check the ‘References’ tab to see if a publication is available, or you can contact us to see if RNA or cell lysates are available for your cell line of interest. Please directly contact us for further information at quotes@abmgood.com.

	Does abm's PriGrow series medium contain antibiotics?
	No, the medium does not contain antibiotics and needs to be supplemented by the end-user as desired.

	Do I need to use abm's media and Applied Cell Extracellular Matrix (G422) to culture my cells?
	We strongly encourage using the recommended media and culture conditions described in the "Growth Conditions" section as they have been optimized for cell growth. Other supplier's media and coating conditions have not been tested.

	What is the difference between Applied Cell Extracellular Matrix (G422) and Collagen Coating?
	The main component of our Applied Cell Extracellular Matrix (G422) is type I collagen specifically. For more information on abm's Applied Cell Extracellular Matrix please visit: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html.

	Do I have to use T25 ECM-coated flasks for growing the cells?
	We strongly recommend thawing cells in the T25 flasks specified on the cell-specific product page to ensure optimal post-thaw recovery of the frozen cells before testing other culture vessels.

	If I want to plate these cells to multi-well plates (e.g. 96 well plates) or dishes, how should I prepare the plates?
	For a protocol on how to coat plates and dishes with Applied Cell Extracellular Matrix (Cat. No. G422), please download the “Applied Cell Extracellular Matrix Data Sheet” from here.

	What percentage of Trypsin-EDTA should be used to subculture cells? Can I use Trypsin-EDTA containing phenol red?
	The standard concentration used for subculturing is 0.25% Trypsin-EDTA, available at abm (Cat. No. TM050). Yes, Trypsin-EDTA containing phenol red can be used as it does not interfere with trypsinization.

	How often do I need to change the media?
	Media should be changed every 2-3 days or as specified within the recommended growth conditions.

Why are these cells classified as biosafety level II?

We follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site, if desired.

	How long can I store frozen vials?
	Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen vapor phase (or below -130°C) indefinitely without affecting viability.

What is the recommended storage temperature for cells?

In general, if you received:

Live cells: acclimatize for 3-5 hours at the recommended temperature and CO2 conditions stated for the cell line under the Growth Conditions section, and then change media afterwards. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).
Frozen cells: Upon receipt, immediately place cells in liquid nitrogen; or below -130°C.

	Can I substitute heat-inactivated FBS with FBS or vice versa?
	FBS and heat-inactivated FBS are different in their composition; they cannot be substituted for one another.

My cells are not detaching, what method do you recommend to trypsinize the cells?

Gently wash adherent cells with PBS (1X, without calcium or magnesium) to remove trypsin inhibitors before adding the trypsinization agent.
Ensure the correct trypsin concentration is being used (i.e. 0.25%).
Do not use cold trypsin to detach cells as the proteolytic activity will be low. Pre-warm trypsin to 37°C for optimal enzymatic activity.
Incubate the cells with trypsin-edta agent at 37°C to facilitate cell detachment.
Gently tap the side of the culture vessel to facilitate mechanical detachment.

How are live cells shipped?

Live cells are shipped as a live culture in a T12.5 flask. Cells are seeded at an optimal confluency to avoid metabolic waste build up during transport. In addition, we thoroughly test our cell lines for functional freeze-thaw recovery. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).

	Why are cells not attaching and forming clumps after subculturing with trypsin?
	Cells form clumps and display attachment issues when the trypsinization agent is not effectively neutralized. To completely neutralize the trypsin agent, add an equal amount of complete growth media prior to centrifugation. Subtle changes in culture conditions, particularly in pH and the quality of serum used in the growth medium, may also affect the amount of clumping exhibited by the cells.

	Why are my cells forming clumps after plating?
	It is important to ensure cell clumps are broken up through gentle re-suspension by pipetting up and down several times after pelleting cells by centrifugation. This will ensure a homogenous single cell suspension for even plating.

	Do I need to add the selection drug to the complete growth medium?
	We recommend maintaining selection pressure using the drug concentration specified in the Growth Conditions section.

	Where can I find the CoA for my product?
	Certificates of analysis can be found here (https://www.abmgood.com/CoA-Library.html).

References

There are no references for this product yet!

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