Isolation of Human Hepatic Sinusoidal Endothelial Cells

Materials and Reagents

1. M199 media

2. Fetal bovine serum (FBS)

3. Hank’s Balanced Salt Solution with calcium and magnesium

4. Petri dish(Ucallm)

5. 50 mland 15ml conical tubes (Ucallm)

6. Tips (Ucallm)

7. Pasteur pipettes (Ucallm)

8. Serological pipettes (Ucallm)

9. Fbronectin coated T25 tissueculture fask(Ucallm)

10. Dispase solution

11. Percoll

12. Culture media for HHSEC, HHSEC were routinely cultured in an endothelial cell selective media, MCDB 131 (GIBCO BRL, Gaithersburg, MD),containing 20% pooled human serum (lrvine Scientifc). The media was also supplemented with the following growth factors and antibiotics: 20 mM L-glutamine (GIBCO), 250 μg/ml cyclicAMP (Sigma), 50 μg/ml epidermal growth factor (Intergen, Purchase, NY), l μg/ml of hydrocortisone (Sigma), 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 250 μg/ml of amphotericin B (GIBCO)

Equipment

1.Centrifuge

2. Cell culture incubator

Procedure

1.Human liver tissue was collected from patients undergoing surgical resection for hepaticmetastasis, Hepaticwarm ischemia time, prior to completion of the resection, was less than 45 min.

2. After removal, liver tissue was thenplaced in Hank’s Balanced Salt Solution with calcium and magnesium(HBSS+; Mediatech, Herndon, VA) at 4 °C.

3. In order to avoid potential con-tamination by large vessel and biliary endothelial cells, identifiable vascularstructures were excised from the liver specimens.

4. The remaining liver tissuewas sharply sectioned into 5-mm cubes, then transferred to a petri dishcontaining 1.2 U/ml of dispase (Boehringer Mannheim, Indianapolis, IN) in Solution A, a N-2-Hydroxyethylpiperazine-N’-2-Ethane Sulfonic Acid (HEPES)buffer (Boehringer), and incubated at 4 °C for 24 h.

5. The tissue cubes werewashed of dispase solution with M199 media (Mediatech) containing 10%fetal bovine serum (FBS; Irvine Scientifc, lrvine, CA).

6. Individual liver cubeswere then mechanically disaggregated in Ml99 media at 4°C, with a flat instrument, to release the endothelial cells. On average, 20-25 cubes were disaggregated in a total of 10 ml of media.

7. The cell suspension was decantedoff, placed into a conical tube, and centrifuged at 4°C for 10 min at 2000 rpm. 

8. After centrifugation, the supernatant was discarded and the cell pelletresuspended in l ml of Ml99 media.

9. The cell suspension was then placedon a density gradient of 35% Percoll (Sigma Chemical Co., St. Louis, MO)and centrifuged at 4 °C for 10 min at 1500 rpm.

10. After centrifugation, thetop band of the gradient (l-2 ml) was removed and mixed with 10 ml ofHBSS+ in a conical tube.

11. This mixture was centrifuged at 4°C, for l0 minat 2000 rpm, the supernatant discarded, and the pellet resuspended in 5 ml of the liver endothelial cell culture media (see materials and reagents).

12. The cell suspensionwas then placed in a fbronectincoated T25 tissue culture fask (Ucallm) and incubated at 37 °C in a 5% CO₂ atmosphere. After 24h, adherent cells were washed with HBSS+ and the media replaced as needed.

References

1.Daneker, G.W., Lund, S.A., Caughman, S.W.et al. Culture and characterization of sinusoidal endothelial cells isolated from human liver. In Vitro Cell.Dev.Biol.-Animal 34, 370–377 (1998).

2.Harrison RL, Boudreau R. Human hepatic sinusoidal endothelial cells in culture produce von Willebrand factor and contain Weibel-Palade bodies. Liver. 1989 Aug;9(4):242-9. doi: 10.1111/j.1600-0676.1989.tb00406.x. PMID: 2788791.

Manufacturer’s Link

Mediatech: https://www.corning.com › emea › keymatch

Boehringer Mannheim: https://www.roche.com.tw>diagnostic-solutions

Irvine Scientifc: https://www.irvinesci.com

Sigma-Aldrich: https://www.sigmaaldrich.com

GIBCO BRL: https://www.thermofisher.com >home>brands >gibco

Intergen: https://www.intergen.com

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