小鼠肝星状细胞Cell Specification
Hepatic stellate cells (HSteC) are pericytes found in the perisinusoidal space and exhibit
myofibroblast-like or lipocyte phenotypes. They participate in the homeostasis, repair, and
regeneration of liver extracellular matrix, and control retinol metabolism, storage, and release.
Following liver injury, HSteC proliferate, transform into myofibroblast-like cells, and produce
type I collagen in the fibrotic liver. HSteC have been implicated as a regulator of hepatic
microcirculation via cell contraction in the pathogenesis of intrahepatic portal hypertension [1].
HSteC also have voltage-activated calcium channels, express the low affinity nerve growth
factor receptor p75, and respond to nerve growth factor stimulation by undergoing apoptosis [2,
3]. Understanding the molecular regulation of HSteC activation may lead to the development of
new therapies for treatment of hepatic fibrosis and chronic liver disease.
MHSteC from ScienCell Research Laboratories are isolated from postnatal day 2 mouse liver.
MHSteC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells
in 1 ml volume. MHSteC are characterized by immunofluorescence with antibodies specific to
小鼠肝星状细胞desmin and α-actin. MHSteC are negative for mycoplasma, bacteria, yeast, and fungi. MHSteC
are guaranteed to further expand for 5 population doublings under the conditions provided by
ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Stellate Cell Medium (SteCM, Cat. #5301) for culturing MHSteC in
vitro.
Product Use
MHSteC 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] Reynaert H, Thompson MG, Thomas T, Geerts A. (2002) “Hepatic stellate cells: role in microcirculation and
pathophysiology of portal hypertension.” Gut 50: 571-81.
[2] Oide H, Tateyama M, Wang XE, Hirose M, Itatsu T, Watanabe S, Ochi R, Sato N. (1999) “Activated stellate
(Ito) cells possess voltage-activated calcium current.” Biochim Biophys Acta. 1418: 158-64.
[3] Trim N, Morgan S, Evans M, Issa R, Fine D, Afford S, Wilkins B, Iredale J. (2000) “Hepatic stellate cells
express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor
stimulation.” Am J Pathol. 156: 1235-43.
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!
Initiating the culture:
1. Prepare a poly-L-lysine-coated culture vessel (2 μg/cm2
, T-75 flask is recommended).
Add 10 ml of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock
solution (10 mg/ml, Cat. #0413). Leave the vessel in a 37oC incubator overnight (or for a
minimum of one hour).
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.
小鼠肝星状细胞3. Rinse the poly-L-lysine-coated vessel twice with sterile water and then add 15 ml of
complete medium. Leave the vessel in the sterile field and proceed to thaw the
cryopreserved cells.
4. Place the frozen vial in a 37
oC 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, poly-L-lysine-coated culture vessel.
A seeding density of 5,000 cells/cm2
is recommended.
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.
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. For best results, 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, then every other day thereafter.
Maintaining the culture:
1. Refresh supplemented culture medium the next morning after establishing a culture from
cryopreserved cells.
2. Change the medium every three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluency, change medium every other day until the
culture is approximately 90% confluent.
Subculturing:
1. Subculture when the culture reaches 90% confluency or above.
2. Prepare poly-L-lysine-coated culture vessels (2 μg/cm2
) one day before subculture.
3. Warm complete medium, trypsin/EDTA solution (T/E, Cat. #0103), 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 37oC water bath
prior to use.
4. Rinse the cells with DPBS.
小鼠肝星状细胞5. Add 8 ml of DPBS and then 2 ml of T/E solution into flask (in the case of a T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask in a 37oC incubator for 1 to 2 minutes or until cells completely round up. Use a
microscope to monitor the change in cell morphology.
6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine
serum (FBS, Cat. #0500).
7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a small percent of cells
may detach) and continue to incubate the flask at 37oC for another 1 to 2 minutes (no
solution in the flask at this moment).
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%.
Note: Use ScienCell T/E solution that is optimized to minimize cell damages due to over
trypsinization.
小鼠肝星状细胞11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 minutes. Resuspend cells in
culture medium.
12. Count and plate cells in a new poly-L-lysine-coated culture vessel with the recommended
cell density.
Caution: Handling animal derived products is potentially biohazardous. 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.