人肝巨噬细胞Cell Specification
Macrophages are cells differentiated from circulating bone marrow-derived monocytes. The main
function of macrophages is to remove cellular debris and destroy invading pathogens. Human
Hepatic Macrophages (HHMa), which are also known as Kupffer cells, reside within the lumen of
liver sinusoids. HHMa protect the liver by responding to pathogens and metastatic cells, while
tolerating harmless self and foreign antigens, which enter via blood flow through the portal vein
and hepatic artery [1]. Recent studies have shown that hepatic macrophages play an important role
in fibrosis, liver inflammation, fatty liver disease, and liver transplantation [2-4]. HHMa are an
excellent model for studying macrophage functions under normal physiological and pathological
conditions.
HHMa from ScienCell Research Laboratories are isolated from human liver. HHMa are
cryopreserved after purification and delivered frozen. Each vial contains > 1 x 106
cells in 1 ml
人肝巨噬细胞volume. HHMa are characterized by immunofluorescence with antibody to F4/80. HHMa are
negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HHMa are guaranteed to
further culture in the conditions provided by ScienCell Research Laboratories; however, HMa
are not recommended for expanding or long term cultures since the cells do not proliferate in
regular culture.
Recommended Medium
It is recommended to use Macrophage Medium (MaM, Cat. #1921) for culturing HHMa in vitro.
Product Use
HHMa are for research use only. They are not approved for human or animal use, or for application
in in vitro diagnostic procedures.
Storage
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.
Shipping
Dry ice.
References
[1] Liaskou E, Wilson DV, Oo YH. (2012) “Innate immune cells in liver inflammation.” Mediators Inflamm. 2012:
949157.
[2] Bieghs V, Verheyen F, van Gorp PJ, Hendrikx T, Wouters K, Lütjohann D, Gijbels MJ, Febbraio M, Binder CJ,
Hofker MH, Shiri-Sverdlov R. (2012) “Internalization of modified lipids by CD36 and SR-A leads to hepatic
inflammation and lysosomal cholesterol storage in Kupffer cells.” PLoS One. 7: e34378.
[3] Tian Y, Jochum W, Georgiev P, Moritz W, Graf R, Clavien PA. (2006) “Kupffer cell-dependent TNF-alpha
signaling mediates injury in the arterialized small-for-size liver transplantation in the mouse.” Proc Natl Acad Sci U
S A. 103: 4598-603.
[4] Seki E, de Minicis S, Inokuchi S, Taura K, Miyai K, van Rooijen N, Schwabe RF, Brenner DA. (2009) “CCR2
promotes hepatic fibrosis in mice.” Hepatology. 50: 185-97.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC water bath and
return the cells to culture as quickly as possible with minimal handling!
Note: Experiments should be well organized before thawing HMa. It is recommended that
HMa are used for experiments as quickly as possible after thawing the cells. HMa cannot be
subcultured or passaged, as the cells do not proliferate.
Initiating the culture:
1. Prepare a poly-L-lysine-coated culture plate (2 μg/cm2
is recommended). For example, add 2
ml of sterile water to one well of a 6-well plate and then add 20l of poly-L-lysine stock
solution (1 mg/ml, Cat. #0403). Leave the plate in a 37oC incubator overnight (or for a
minimum of one hour).
2. Prepare complete medium (MaM, Cat. #1921). Thaw MaGS (Cat. #1972), FBS (Cat. #0025)
and P/S solution (Cat. #0503) at 37oC. Gently tilt the tubes several times to ensure the contents
are completely mixed before adding to the medium. Decontaminate the external surfaces of
medium bottle and medium supplement tubes with 70% ethanol and transfer them to a sterile
field. In a sterile field, remove the caps without touching the interior threads with fingers. Add
人肝巨噬细胞MaGS, FBS and P/S solution to the medium and mix well.
3. Rinse the poly-L-lysine-coated vessel twice with sterile water and then add the volume of
complete medium recommended in Table 1 or Table 2. Leave the plate(s) in the sterile field
and proceed to thaw the cryopreserved cells.
4. Place the frozen vial in a 37oC 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.
Note: Dilution and centrifugation of cells after thawing are not recommended since 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 poly lysine coated culture vessels to promote cell
attachment.
5. Carefully remove the cap without touching the interior threads and gently resuspend the cell
suspension. A seeding density of 10,000-20,000 cells/cm2
is recommended depending on your
experiments. We recommend following Table 1 for seeding HHMa onto 6-well, 12-well, or
24-well plates. For seeding HHMa on 60 mm plates, use Table 2.
Table 1
Recommended cell suspension volume per vial using a 6-well, 12-well, or 24 well format
Well format Surface area/well
(approx. values)
Volume of media/well Volume of cell suspension
from vial/well
# of wells/vial
6-well 9.6 cm2 3.0 ml 150 l 6 wells
12-well 3.9 cm2 2.0 ml 60 l 15 wells
24-well 1.9 cm2 1.0 ml 30 l 30 wells
Table 2
Recommended cell suspension volume per vial using 60 mm plates
Plate Format Surface area/plate
(approx. values)
Volume of cell
suspension from
vial/plate
# of plates/vial Volume of media
(ml)/plate
60 mm 21 cm2
300 l 3 3.0 ml
6. Pipet the correct volume of cell suspension into each well of an equilibrated, poly-L-lysinecoated
culture plate containing complete medium. Replace the lid of the culture plate and
gently rock the plate to distribute the cells evenly.
7. Return the culture plate to the incubator.
8. For best results, do not disturb the culture for at least 16 hours after the culture has been
initiated. Change the culture medium the next morning after establishing a culture from
cryopreserved cells to remove residual DMSO and unattached cells. Once the macrophages
attach, the cells can be used for experiments.
9. Use cells promptly for experiments.
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%
人肝巨噬细胞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 [1].
[1] 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.