人冠状动脉内皮细胞Cell Specification
Endothelial cells constitute the natural interface between the blood and the underlying tissue. Changes in
endothelial cell function appear to play a key role in the pathogenesis of atherosclerosis. Endothelial cells
synthesize and secrete activators as well as inhibitors of both the coagulation system and the fibrinolysis
system in addition to mediators that influence the adhesion and aggregation of blood platelets. Endothelial
cells also release molecules that control cell proliferation and modulate vessel wall tone [1]. Human
endothelial cells produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to
TNF-alpha by modifying growth characteristics, producing cytokines such as GM-CSF, expressing
ICAM-1 on the surface and producing large amounts of nitric oxide and endothelin [2]. Endothelial
injury, with consequent endothelial dysfunction, is caused by percutaneous transluminal coronary
angioplasty [3] and may play an important role in subsequent restenosis [4]. Many of the endothelial
processes can be studied in vitro using cultured cells, and HCAEC will surely be very useful to studying
and understanding the molecular mechanisms involved in coronary artery diseases.
HCAEC from ScienCell Research Laboratories are isolated from human coronary artery. HCAEC are
cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml volume.
HCAEC are characterized by immunofluorescent method with antibodies to vWF/Factor VIII and CD31
(P-CAM) and by uptake of DiI-Ac-LDL. HCAEC are negative for HIV-1, HBV, HCV, mycoplasma,
bacteria, yeast and fungi. HCAEC are guaranteed to further expand for 15 population doublings at the
conditions provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Endothelial Cell Medium (ECM, Cat. No. 1001) for the culturing of HCAEC in
vitro.
Product Use
HCAEC are for research use only. It is not approved for human or animal use, or for application in in
vitro diagnostic procedures.
Storage
Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep the cells
in liquid nitrogen until cell culture needed for experiments.
Shipping
Dry ice.
Reference
[1] Elhadj, S. and Forsten, K. E. (2001) Chronic shear stress affects bovine aortic endothelial cell secreted proteoglycan
production in vitro. Bioengineering Conference, Vol. 50:767-768.
[2] Donnini, D., Perrella, G., Stel, G., Ambesi-Impiombato, F. S., Curcio, F. (2000) A new model of human aortic endothelial
cells in vitro. Biochimie 82:1107-14.
人冠状动脉内皮细胞[3] Vassanelli C, Menegatti G, Zanolla L, Molinari J, Zanotto G, Zardini P. (1994) Coronary vasoconstriction in response to
acetylcholine after balloon angioplasty: possible role of endothelial dysfunction. Coron Artery Dis. 5:979-986.
[4] Meurice T, Vallet B, Bauters C, Dupuis B, Lablanche JM, Bertrand ME. (1996) Role of endothelial cells in restenosis after
coronary angioplasty. Fundam Clin Pharmacol. 10:234-242.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
C waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the ordering:
1. Prepare a fibronectin coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml of
sterile Dulbecco’s phosphate buffered saline (DPBS, Ca++ and Mg++ free, cat. no. 0303)
to a T-75 flask and then add 150 μl of fibronectin stock solution (1 mg/ml, Sigma cat. no.
F1141). Leave the flask in incubator overnight.
2. Prepare complete medium: decontaminate the external surfaces of medium and medium
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.
3. Aspirate fibronectin solution and add 20 ml of complete medium to the flask. Leave the
flask in the hood and go to thaw the cells. The fibronectin solution can be used twice.
4. Place the vial in a 37o
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.
5. Dispense the contents of the vial into the equilibrated, fibronectin coated culture vessels.
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 DMSO residue in the culture. It is
also important that endothelial cells are plated in fibronectin coated flask that promotes
cell attachment and growth.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
人冠状动脉内皮细胞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. A healthy culture will display
cobblestone or spindle shaped morphology, non-granular cytoplasm and the cell number
will be double after two to three days in culture.
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 fibronectin coated flasks (2 μg/cm2
) one day before subculture.
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS to room temperature. We do not recommend
warming the reagents and medium at 37o
C waterbath prior to use.
4. Rinse the cells with DPBS.
5. Add 10 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
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
C for 1 minutes (no solution in
the flask at this moment); at the end of trypsinisation, 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%.
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
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, fibronectin coated flask with cell density as
recommended.
人冠状动脉内皮细胞Caution: Handling human derived products is potentially bioharzadous. Although each cell strain testes negative
for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions
mush be taken to avoid inadvertent exposure. 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).