大鼠肝脏星形胶质细胞Cell Specification
Hepatic stellate cells (HSteC) are intralobular connective tissue cells presenting myofibroblastlike
or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix,
repair, regeneration, fibrosis and control retinol metabolism, storage and release. Following liver
injury, HSteC transform into myofibroblast-like cells and are the major source of type I collagen
in the fibrotic liver. Beyond these feature, HSteC have been implicated as regulators of hepatic
microcirculation via cell contraction, and in disease states, in the pathogenesis of intrahepatic
portal hypertension [1, 2]. Proliferation and migration of HSteC and expression of chemokines
are involved in the pathogenesis of liver inflammation and fibrogenesis. HSteC possess voltageactivated
calcium current, express the low affinity nerve growth factor receptor p75, and undergo
apoptosis in response to nerve growth factor stimulation [3, 4]. Therefore, the new insight into
the molecular regulation of HSteC activation will lead to therapeutic approaches in treatment of
hepatic fibrosis in the future, and could lead to reduced morbidity and mortality in patients with
chronic liver injury.
RHSteC from ScienCell Research Laboratories are isolated from neonate day 2 rat liver tissue.
RHSteC are cryopreserved immediately after purification and delivered frozen. Each vial
contains >5 x 105
Recommended Medium
cells in 1 ml volume. RHSteC are characterized by immunofluorescent method
with antibodies to desmin and α-actin. RHSteC are negative for mycoplasma, bacteria, yeast and
fungi. RHSteC are guaranteed to further expand for 5 population doublings in the conditions
provided by ScienCell Research Laboratories.
It is recommended to use Satellite Cell Medium (SCM, Cat. No.5301) for the culturing of
RHSteC in vitro.
Product Use
大鼠肝脏星形胶质细胞RHSteC are for research use only
Storage
. It is not approved for human or animal use, or for application
in in vitro diagnostic procedures.
Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep
the cells in liquid nitrogen until cell culture is needed for experiments.
Shipping
Dry ice.
Reference
[1] Reynaert H, Thompson MG, Thomas T, Geerts A. (2002) Hepatic stellate cells: role in microcirculation and
pathophysiology of portal hypertension. Gut 50:571-581.
[2] Rockey D. C. (2001) Hepatic blood flow regulation by stellate cells in normal and injured liver. Semin Liver Dis
21(3):337-49.
[3] 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. 1418:158-164.
[4] 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(4):1235-1243.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
and return them to culture as quickly as possible with minimal handling!
C waterbath
Set up culture after receiving the order:
1. Prepare a poly-L-lysine coated flask (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, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37o
2. Prepare complete medium: decontaminate the external surfaces of medium and medium
大鼠肝脏星形胶质细胞supplements with 70% ethanol and transfer them to sterile field. Aseptically open each
supplement tube and add them to the basal medium with a pipette. Rinse each tube with
medium to recover the entire volume.
C incubator).
3. Rinse the poly-L-lysine coated flask with sterile water twice and add 20 ml of complete
medium to the flask. Leave the flask in the hood and go to thaw the cells.
4. Place the vial in a 37o
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of 5,000 cells/cm
C waterbath, hold and rotate the vial gently until the contents are
completely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse
the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful
not to touch the interior threads with fingers. Using a 1 ml eppendorf pipette gently resuspend
the contents of the vial.
2
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of DMSO residue in the culture. It is
also important that cells are plated in poly lysine coated flask that promotes cell
attachment and growth.
is recommended.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
cap if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. For best result, do not disturb the culture for at least 16 hours after the culture has been
initiated. Change the growth medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter.
Maintenance of Culture:
1. Change the medium to fresh supplemented 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% confluence, change medium every other day until the
culture is approximately 90% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS (Ca
).
++ and Mg++ free, cat. no. 0303) to room
temperature. We do not recommend warming the reagents and medium at 37o
4. Rinse the cells with DPBS.
C
waterbath prior to use.
5. Add 8 ml of DPBS first and then 2 ml of trypsin/EDTA solution into flask (in the case of
T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37o
大鼠肝脏星形胶质细胞C incubator for 1 to 2 minutes or until cells are
completely rounded up (monitored with inverted microscope). During incubation, prepare
a 50 ml conical centrifuge tube with 5 ml of fetal bovine serum (FBS, cat. no. 0500);
transfer trypsin/EDTA solution from the flask to the 50 ml centrifuge tube (a few percent
of cells may detached); continue incubate the flask at 37o
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
C for 1 or 2 minutes more (no
solution in the flask at this moment); at the end of trypsinization, one hand hold one side
of flask and the other hand gently tap the other side of the flask to detach cells from
attachment; check the flask under inverted microscope to make sure all cells are
detached, add 5 ml of trypsin neutralization solution to the flask and transfer detached
cells to the 50 ml centrifuge tube; add another 5 ml of TNS to harvest the residue cells
and transfer it to the 50 ml centrifuge tube. Examine the flask under inverted microscope
to make sure the cell harvesting is successful by looking at the number of cells left
behind. There should be less than 5%.
6. Centrifuge the 50 ml centrifuge tube (harvested cell suspension) at 1000 rpm (Beckman
Coulter Allegra 6R centrifuge or similar) for 5 min; re-suspend cells in growth medium.
7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
Caution: Handling animal derived products is potentially biohazardous. Always wear gloves and safety glasses
when working 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, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues. J Tissue Culture Methods. 11(4).