Primary Human Hepatic Sinusoidal Endothelial Cells Culture Protocol

• What are hepatic Sinusoidal Endothelial Cells?
• Hepatic Sinusoidal Endothelial Cells Culture Materials
• Hepatic Sinusoidal Endothelial Cell Culture Method

What are hepatic Sinusoidal Endothelial Cells (HSECs)?

Human hepatic Sinusoidal Endothelial cells (HSECs) are specialized endothelial cells in the liver that participate in the receptor-mediated clearance of endotoxin, bacteria, and other compounds. HSECs are also known to regulate inflammation, leukocyte recruitment, and host-immune responses to invading pathogens. Human HSECs are a valuable tool for the study of liver physiology and pathophysiology in vitro.

Our collection of hepatic Sinusoidal Endothelial cells is isolated through collagenase digestion and selective cell culture media. Our frozen HSECs are cryopreserved at the end of the primary culture. Each lot undergoes testing for specific cell markers and comes with a guarantee of ≥70% post-thaw viability. In this protocol, we demonstrate how to thaw, plate, and culture primary human hepatic Sinusoidal Endothelial cells.

Primary Sinusoidal Endothelial Cells Culture Materials 

• Normal Human Sinusoidal Endothelial Cells (SECs)
  Note: Upon receipt, immediately store cryovial(s) in vapor phase liquid nitrogen. 
• Collagen Type I, Rat Tail
• Tissue culture treated multiwell plates
• Dulbecco’s PBS, without Calcium & Magnesium (DPBS)
• Trypsin Solution
• Human Sinusoidal Endothelial Cell Maintenance Medium¹:

** Available from Gibco.

Components	Catalog Number	Working Stock	Final Dilution	Final Conc.	Final Volume (mL)
Human Endothelial Medium	11111044**			1x	450
AB Human Serum	H3667-100ML			10%	50
Human VEGF	SRP3182-10UG	0.1mg/mL	10,000x	10ng/mL	0.05
Human HGF	H5791-10UG	0.1mg/mL	10,000x	10ng/mL	0.05
				Total volume	500

Human Sinusoidal Endothelial Cell Culture Method

The following protocols were performed using a Class II laminar flow biohood and an aspirator. All incubators used were set to 37°C and 5% CO₂. PPE should be worn at all times, including safety glasses, lab coats, and gloves.

Collagen-Coated Plate Preparation Protocol

1. Dilute the collagen to a final concentration of 56µg/mL in sterile 70% ethanol. Gently mix the solution until the collagen is fully solubilized.
2. Completely cover the bottom of well with the collagen/ethanol mixture.
3. Evenly coat the inside of each well with the collagen/ethanol mixture by moving the cell culture plate gently.
4. Allow plates to air dry in a laminar flow hood. Leave cell culture plate over night with the cover ajar to allow airflow and prevent condensation.

Thawing and Plating Human Hepatic Sinusoidal Endothelial Cells 

1. Thaw vial in a 37˚C water bath, holding and rotating it gently. Once thawed, remove the vial from the water bath, wipe dry, rinse it with 70% ethanol and transfer to sterile field. Remove the cap, do not touch the interior thread with your fingers.
2. Transfer the contents of the vial to a 15ml conical tube using a pipette. Using 5ml of medium, wash the vial and transfer to the conical tube.
3. Centrifuge the conical tube at 300 x g for 5 minutes. After this centrifugation, aspirate the medium and resuspend the contents in the medium.
4. Count the resulting cells using a cell counter like the Scepter 3.0 Handheld Automated Cell Counter.
5. For expansion, seed the HHSECs at a density of 5,000 cells/cm² on Collagen I coated plates (see above protocol).
6. For best results, do not disturb the HHSEC culture for at least 12 hours after initial seeding. To remove any residual DMSO or unattached cells, change the growth medium the next day.
7. Continue to change media every other day until ready for use.

Sub-Culturing Human Hepatic Sinusoidal Endothelial Cells 

1. Subculture cells when they have reached 70-80% confluency. Cell confluency and health can be monitored using the Millicel^® DCI Digital Cell Imager.
2. Warm the HHSEC medium, Dulbecco’s Phosphate Buffered Saline, without Calcium & Magnesium (DPBS), and 0.25% trypsin solution to room temperature.
3. Aspirate the medium, then rinse the HHSEC culture with DPBS. Add the trypsin solution to the flask and place in a 37°C incubator for 3-5 minutes or until the cells fully detach.
4. Once cells have detached, collect HHSECs from the culture flask with an appropriate amount of medium.
5. Transfer the HHSECs to a centrifuge tube and centrifuge at 300 x g at room temperature for 5 minutes.
6. After centrifugation aspirate the medium, re-suspend the pellet, and count cells for seeding.
7. Seed the cells to 5,000 cells/cm² on collagen I coated plates (see above protocol).

References

1. Lalor PF, Edwards S, McNab G, Salmi M, Jalkanen S, Adams DH. 2002. Vascular Adhesion Protein-1 Mediates Adhesion and Transmigration of Lymphocytes on Human Hepatic Endothelial Cells. 169(2):983-992. https://doi.org/10.4049/jimmunol.169.2.983