|文件名稱：||sciencellonline 5000 HHSEC|
sciencellonline 5000 HHSEC
Human Hepatic Sinusoidal Endothelial Cells
Endothelial cells perform multiple physiological functions and are central to many pathological
processes. The liver contains two distinct endothelial cell types: vascular and sinusoidal. Sinusoidal
endothelial cells (SEC) are microvascular endothelial cells with a unique phenotype reminiscent of
dendritic cells and a unique function as antigen-presenting cells for CD4+ T cells. Thus, SEC
represent a new type of organ-resident "non-professional" antigen-presenting cell that appears to be
involved in the local control of the immune response and the induction of immune tolerance in the
liver . The hepatic microenvironment, including the portal venous constituents and soluble
mediators from sinusoidal cell populations, tightly control antigen presentation by SEC to avoid
immune-mediated damage. SEC express well-characterized surface receptors and differ
morphologically and metabolically from large-vessel endothelia . Previous studies have shown
that SEC are dynamic regulators of porosity that respond rapidly and locally to environmental zonal
stimuli during liver regeneration . SEC dysfunction and structural alterations have far-reaching
repercussions for the whole liver.
HHSEC from ScienCell Research Laboratories are isolated from human liver. HHSEC are
cryopreserved immediately after purification and delivered frozen. Each vial contains >5 x 105 cells
in 1 ml volume. HHSEC are characterized by immunofluorescence with antibodies specific to
vWF/Factor VIII and CD31 (PECAM). HHSEC are negative for HIV-1, HBV, HCV, mycoplasma,
bacteria, yeast and fungi. HHSEC are guaranteed to further expand for 5 population doublings
under the conditions provided by ScienCell Research Laboratories. Note: HHSEC are not expected
to proliferate many times in culture, so we recommend using cells for experiments at earliest
passage after initial plating.
It is recommended to use Endothelial Cell Medium (ECM, Cat. #1001) for the culturing of HHSEC.
HHSEC are for research use only. It is not approved for human or animal use, or for application in
in vitro diagnostic procedures.
Upon receiving, directly and immediately transfer the cells from dry ice to liquid nitrogen and keep
the cells in liquid nitrogen until they are needed for experiments.
 Limmer A, Knolle PA. (2001) “Liver sinusoidal endothelial cells: a new type of organ-resident antigen-presenting
cell.” Arch Immunol Ther Exp (Warsz) 49 Suppl 1:S7-11.
 Wack KE, Ross MA, Zegarra V, Sysko LR, Watkins SC, Stolz DB. (2001) “Sinusoidal ultrastructure evalsuated
during the revascularization of regenerating rat liver.” Hepatology 33(2):363-78.
 Gervaz P, Scholl B, Mainguene C, Poitry S, Gillet M, Wexner S. (2000) “Angiogenesis of liver metastases: role of
sinusoidal endothelial cells.” Dis Colon Rectum 43(7):980-6.Instructions for culturing primary cells
Cryopreserved primary cells are very delicate. Thaw the vial in a 37o C water bath
and return the cells to culture as quickly as possible with minimal handling! Do
not centrifuge the cells after thawing as this can damage the cells.
Note: HHSEC are very sensitive cells and they are not expected to proliferate many times
in culture. Experiments should be well organized before thawing the cells. It is
recommended that HHSEC are used for experiments at earliest passage after initial plating
with minimal expansion. If subculture is inevitable, follow the instructions below with
Initiating the culture:
Note: ScienCell primary cells must be cultured in a 37o C, 5% CO2 incubator. Cells are only
warranted if ScienCell media and reagents are used and the recommended protocols are followed.
1. Prepare a fibronectin-coated culture vessel (2 µg/cm2 , T-75 flask is recommended). To
obtain a 2 µg/cm 2 fibronectin-coated culture vessel, add 5 ml of sterile Dulbecco’s phosphate
buffered saline, Ca++- and Mg++-free (Cat. #0303) to a T-75 flask and then add 150 µl of
fibronectin stock solution (Cat. #8248). Leave vessel in a 37o C incubator overnight (or for at
least 2 hours).
2. Prepare complete medium. Decontaminate the external surfaces of medium bottle and
medium supplement tubes with 70% ethanol and transfer them to a sterile field. Aseptically
transfer supplement to the basal medium with a pipette. Rinse the supplement tube with
medium to recover the entire volume.
3. Aspirate the fibronectin solution and add 15 ml of complete medium to the culture vessel.
The fibronectin solution can be reused twice. Leave the vessel in the sterile field and
proceed to thaw the cryopreserved cells.
4. Place the frozen vial in a 37o C water bath. Hold and rotate the vial gently until the contents
completely thaw. Promptly remove the vial from the water bath, wipe it down with 70%
ethanol, and transfer it to the sterile field.
5. Carefully remove the cap without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, fibronectin-coated culture vessel.
Note: Dilution and centrifugation of cells after thawing are not recommended as these
actions are more harmful to the cells than the effect of residual DMSO in the culture. It is
also important that cells are plated in fibronectin-coated culture vessels to promote cell
6. Replace the cap or lid of the culture vessel and gently rock the vessel to distribute the cells
evenly. Loosen cap, if necessary, to allow gas exchange.
7. Return the culture vessel to the incubator.
8. Do not disturb the culture for at least 16 hours after the culture has been initiated. Refresh
culture medium the next day to remove residual DMSO and unattached cells.
Maintaining the culture:1. Refresh supplemented culture medium the next morning after establishing a culture from
2. Change the medium every three days thereafter, until the culture is approximately 70%
3. Once the culture reaches 70% confluency, change medium every other day until the culture
is approximately 90% confluent.
1. Subculture when the culture reaches 90% confluency.
2. Prepare fibronectin-coated culture vessels (2 µg/cm2) one day before subculture.
3. Warm complete medium, trypsin/EDTA solution, 0.05% (T/E, Cat. #0183), T/E
neutralization solution (TNS, Cat. #0113), and DPBS (Ca++- and Mg++-free, Cat. #0303) to
room temperature. We do not recommend warming reagents and medium in a 37o C water
bath prior to use.
4. Rinse the cells with DPBS.
5. Add 8 ml DPBS and 2 ml 0.05% T/E solution (Cat. #0183) into flask (in the case of a T-75
flask). Gently rock the flask to ensure complete coverage of cells by T/E solution. Use a
microscope to monitor the change in cell morphology.
Note: We recommend using ScienCell’s 0.05% T/E solution, which is optimized to
minimize cell damage due to over trypsinization. If 0.25% T/E solution (Cat. #0103) is
used, then 9.6 ml of DPBS and 0.4 ml of 0.25% T/E solution should be used.
Caution: Do NOT use undiluted trypsin when subculturing primary cells.
6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine serum
(FBS, Cat. #0500).
7. Once the cells completely round up, transfer T/E solution from the flask to a 50 ml
centrifuge tube (a small percent of cells may detach) and continue to incubate the flask at
37o C for another minute (no solution in the flask at this time).
8. At the end of incubation, gently tap the side of the flask to dislodge cells from the surface.
Check under a microscope to make sure that all cells detach.
9. Add 5 ml of TNS solution to the flask and transfer detached cells to the 50 ml centrifuge
tube. Rinse the flask with another 5 ml of TNS to collect the residual cells.
10. Examine the flask under a microscope for a successful cell harvest by looking at the number
of cells being left behind; there should be less than 5%.
11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 minutes. Gently resuspend cells in
12. Count and plate cells in a new fibronectin-coated culture vessel with the recommended cell
density. A seeding density of 6,000-8,000 cells/cm2 is recommended.
Note: We do not recommend cryopreservation of primary cells by the end user. Refreezing
cells may damage them and affect cell performance. ScienCell does not guarantee primary
cells cryopreserved by the end user. Caution: Handling human derived products is potentially biohazardous. Although each cell strain tests negative for
HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions must
be taken to avoid inadvertent exposure. Always wear gloves and safety glasses when working with these materials.
Never mouth pipette. We recommend following the universal procedures for handling products of human origin as
the minimum precaution against contamination .
 Grizzle WE, Polt S. (1988) “Guidelines to avoid personal contamination by infective agents in research laboratories
that use human tissues.” J Tissue Cult Methods. 11: 191-9.