人肾上腺皮质细胞Cell Specification
The adrenal gland plays an essential role in regulating homeostasis in the body through the
secretion of corticosteroid and androgen hormones. The secreted steroids arise from three zones
that form the adrenal gland: the zona glomerulosa, zona fasciculate, and zona reticularis.
Microvascular endothelial cells line the blood vessels and contribute to many biological
processes such as hemostasis, coagulation, trafficking of lymphocytes, and the inflammatory
response. Microvascular endothelial cells (MEC) are of importance in the adrenal gland for their
regulatory function on the secretion of adrenal hormones [1]. Studies have shown that factors
secreted from HAdMEC, such as endothelin-1, nitric oxide, adrenomedullin and PAMP affect
aldosterone secretion [2]. HAdMEC differ from MECs from different organs in function and
phenotype [3]. For example, compared to brain MEC and liver MEC, adrenal MEC are highly
resistant to glucocorticoid-induced cytotoxicity showing that MECs are specialized to the tissues
they belong to [4].
HAdMEC from ScienCell Research Laboratories are isolated from human adrenal tissue.
HAdMEC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 105
cells in 1 ml volume. HAdMEC are characterized by immunofluorescent method with antibodies
to vWF/Factor VIII and CED31 (P-CAM). HAdMEC are negative for HIV-1, HBV, HCV,
人肾上腺皮质细胞mycoplasma, bacteria, yeast and fungi. HAdMEC are guaranteed to further culture for 15
population doublings in 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
HAdMEC in vitro.
Product Use
HAdMEC 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][2] Hinson, J.P., Kapas, S. 1998. The role of endothelial cell product in the regulation of adrenocortical functions:
actions of endothelin, nitric oxide, adrenomedullin and PAMP. Horm Metab Res. 30(6-7): 334-40
[3] Gu, F., Zhang, WJ., Wu, LQ., Ye, LY., Li, HZ., Li, J., Xu, SQ., XU, YP., Lou, JN. 2006. Characteristics of
human adrenal microvascular endothelial cells in morphology, phenotypes, and function. Ahonqquo Yi Xue Ke
Xue Yuan Xue Bao. 28(4):502-6
ScienCell
Research Laboratories
TM
人肾上腺皮质细胞Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the order:
1. Prepare a fibronectin coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml of
sterile Dulbecco’s phosphate buffered saline, Ca++ and Mg++ free (ScienCell, Cat. No.
0303) to a T-75 flask and then add 150 μl of fibronectin stock solution (ScienCell, Cat.
No. 8248). 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 37oC 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 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
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.
人肾上腺皮质细胞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 37oC 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 37oC 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 37oC for 1 minutes (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%.
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.
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 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).